In Vitro Cell.Dev.Biol.

Plant (2012) 48:153159

DOI 10.1007/s11627-011-9421-0

BIOTECHNOLOGY

A rapid and efficient method for in vitro shoot organogenesis

and production of transgenic Bacopa monnieri L. mediated
by Agrobacterium tumefaciens
Aileni Mahender & Bulle Mallesham & Kota Srinivas &
Gadidasu Kranthi Kumar & Kokkirala Venugopal Rao &
Yarra Rajesh & Peng Zhang & Abbagani Sadanandam

Received: 29 June 2011 / Accepted: 4 December 2011 / Published online: 25 January 2012 / Editor: John W. Forster
# The Society for In Vitro Biology 2012

Abstract Efficient shoot regeneration and Agrobacteriummediated genetic transformation systems were developed for
Bacopa monnieri L. (Scrophulariaceae), a plant well known
for its medicinal properties. Leaf explants were cultured on
Murashige and Skoog (MS) medium with different concentrations of 6-benzylaminopurine (BAP), and in combination
with either indole-3-acetic acid (IAA) or napthalene-3-acetic
acid. A combination of BAP (17.80 M) and IAA (2.28 M)
maximized shoot initiation (85.22.43) with greatest shoot
length (2.8 0.22), and was obtained directly from leaf
explants without an intervening callus phase. Leaf segments
from in vitro grown plants were co-cultivated with Agrobacterium tumefaciens LBA4404 harboring pCAMBIA1301
with -glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes. The co-cultivated explants were transferred
to selective shoot induction and elongation medium. The
elongated hygromycin-resistant shoots were subsequently
rooted on MS medium supplemented with 4.9 M indole-3butyric acid and 25 mg/l hygromycin (SSRM). Successful
transformation was confirmed by monitoring histochemical
GUS activity during shoot elongation and PCR analyses using
uidA- and hpt-specific primers. Integration of hpt into the
genome of transgenic plants was also verified by Southern
A. Mahender : B. Mallesham : K. Srinivas : G. Kranthi Kumar :
K. Venugopal Rao : Y. Rajesh : A. Sadanandam (*)
Plant Biotechnology Research Unit, Department of Biotechnology,
Kakatiya University,
Warangal 506 009, India
e-mail: nandamas@rediffmail.com
A. Mahender : G. Kranthi Kumar : P. Zhang
National Key Laboratory of Plant Molecular Genetics, Institute of
Plant Physiology and Ecology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences,
300 Fenglin Road,
Shanghai 200032, China

blot analysis. The highest transformation efficiency achieved

was 70.6%, with an average of 10.40.15 transgenic plantlets
per explant using the present transformation system. Therefore, these highly efficient and rapid regeneration and transformation systems create significant potential for engineering
of B. monnieri with a view to detailed biomolecular analyses
or for further enhancement of its medicinal properties.
Keywords Bacopa monnieri . Genetic transformation .
Organogenesis

Introduction
Indian medicine folklore purports that certain herbs can be
used as traditional brain or nerve tonics. One of the most
popular is Bacopa monnieri L. (Scrophulariaceae), commonly
called Brahmi, a small, amphibious plant growing in marshy
areas throughout the Indian subcontinent. Brahmi is also
known as Medhya Rasayana in Ayurveda as it has been
reported to increase mental clarity and brain-stimulating activities (Bhattacharya and Ghosal 1998). It also possesses properties to protect against inflammation, epilepsy, insanity,
cancer as well as offering analgesic, antipyretic, and antioxidant activities (Jain et al. 1994; Elangovan et al. 1995; Tripathi
et al. 1996; Vohora et al. 1997).
The medicinal properties of Brahmi are attributed to the
presence of different types of saponins such as bacosides A, B,
C, and D which are biologically active triterpenoids, more
commonly known as memory chemicals (Rastogi et al.
1994). Bacopa extract has anxiolytic, cognition-enhancing
(Bhattacharya and Ghosal 1998), relaxing (Dar and Channa
1997), antioxidant (Mukherjee and Dey 1966), and immunomodulator activities (Dahanukar and Thatte 1997). The

154

MAHENDER ET AL.

(IAA) in inducing shoot regeneration in B. monnieri was

studied, and was used to successfully develop a simple,
rapid, and efficient Agrobacterium tumefaciens-mediated
genetic transformation protocol for this important medicinal
plant species.

natural regeneration of this herb is hampered due to short

viability (2 mo) of seeds, frequent death of seedlings at a
two-leaf stage, and strict habitat requirements for marshy
areas.
In comparison to the abundant pharmacological data,
clinical studies and reports on in vitro plant regeneration
systems (Shrivastava and Rajani 1999; Vaibhav et al. 2001),
including genetic engineering of B. monnieri, are sparse
(Nisha et al. 2003; Ramesh et al. 2011). Development of a
highly efficient plant regeneration system is a prerequisite
for production of transgenic plants by Agrobacteriummediated transformation, and an essential prerequisite for
genetic improvement of this valuable medicinal plant species.
Recently, various biotechnological approaches have been
adopted for increasing the level of bioactive molecules in
medicinal plants or microbes (Grotewold 2008; Schafer and
Wink 2009). Through genetic manipulation of biosynthetic
pathways via genetic transformation, success has been
reported in enhancing the production of secondary metabolites in some species (Rosatil et al. 2000; Ye et al. 2000;
Mahmoud and Croteau 2001; Goossens and Rischer 2007;
Oksman-Caldentey et al. 2007; Palazon et al. 2008; Liu et
al. 2010; Yang et al. 2011). Developing protocols for efficient genetic transformation of medicinal plants is an important tool to study the molecular basis and regulation of
metabolic pathways (Khan et al. 2009). It is also an essential
knowledge base to move from empirical to predictive metabolic engineering (Dixon 2005). In the present study, the
role of 6-benzylaminopurine (BAP) and indole-3-acetic acid

Table 1. The effects of BAP or

BAP+IAA, and BAP+NAA
on regeneration from leaf
explants in B. monnieri L.

In each column, mean followed

by same lowercase letter was
not significantly different
(p0.01) according to Duncans
multiple test
S shoot, C callus
z
The values are means of 40
explants SE

Growth regulator (M)

BAP
8.90
17.80
26.70
BAP + IAA
8.90+1.14
17.80+1.14
26.70+1.14
8.90+2.28
17.80+2.28
26.70+2.28
BAP+NAA
8.90+1.14
17.80+1.14
26.70+1.40
8.90+2.28
17.80+2.28
26.70+2.28

Materials and Methods

Shoot organogenesis via leaf explants. Plant material was
collected from medicinal arboretum maintained by the Forest Department, Warangal, India. Leaves from 3-month-old
field-grown mature B. monnieri L. plants were thoroughly
washed under running tap water and placed in 5% Tween 20
for 5 min, followed by three to four rinses in sterile distilled
water. Further sterilization was performed under aseptic
conditions inside a laminar flow cabinet by immersing the
leaf material in an aqueous solution of 0.1% HgCl2 for 4
5 min, followed by four to five rinses in sterile distilled
water. Leaf tissue was cut into 1.0-cm squares and cultured
in 150 25-cm Borosil tubes each containing 20 ml of
Murashige and Skoog (MS) medium (Murashige and Skoog
1962), supplemented with 2% w/v sucrose, 0.8% w/v agar,
and BAP (8.90, 17.80 or 26.70 M) alone, or in combination with IAA (1.14 or 2.28 M) or napthalene-3-acetic acid
(NAA; 1.14 or 2.28 M; Table 1). After 2530 d on the
regeneration medium, leaf cultures were transferred onto MS
basal medium (MS+2% w/v sucrose, 0.8% w/v agar). After
1 wk, elongated shoots (45 cm) were excised and transferred

Response (%)

Morphogenic response
(shootcallus)

Shoots
(no./explant)z

Shoot length (cm)z

60
80
55

S+C
S+C
S+C

35.50.26 a
44.20.41 a
40.10.07 a

1.60.40 a
2.21.32 b
1.80.26 a

65
85
70
70
90
60

S
S
S
S
S
S

55.10.12 b
75.70.43 c
60.40.35 c
58.51.30 b
85.22.43 c
66.80.26 c

2.01.30
2.60.21
2.40.43
2.21.14
2.80.22
2.50.45

55
70

S+C
S+C

45.21.19 a
60.60.26 c

1.80.26 a
2.01.40 b

60
40
60
50

S+C
S+C
S+C
S+C

54.40.61 b
40.12.31 a
55.70.12 b
40.40.69 a

2.20.69
1.70.60
2.00.32
1.80.12

b
d
c
b
d
c

b
a
b
a

SHOOT ORGANOGENESIS AND PRODUCTION OF TRANSGENIC BACOPA

to rooting to culture tubes containing MS medium with

indole-3-butyric acid (IBA; 2.4 or 4.9 M). All experiments
were carried out in MS medium with 2% w/v sucrose and
0.8% w/v agar (Himedia, India), with the pH of the media
adjusted to 5.8 using 0.1 N NaOH before autoclaving (121C
for 15 min). Each experiment was repeated thrice, with two
replicates per experiment. Each experiment included 50 (25+
25) explants for further analysis. All cultures were maintained
at 252C under white fluorescent light (65 E/m2/s) with a
16-h photoperiod.
In vitro-generated rooted shoots with seven to eight
leaves were removed from culture tubes and washed gently
under running tap water to remove excess medium before
transfer to plastic pots containing an equal mixture of vermiculite and perlite (1:1). The potted plantlets were watered
and hardened in the greenhouse (28C day, 24C night at
65% relative humidity) for 810 d.
Data pertaining to responding cultures, percent of
explants exhibiting shoot development, the number and
length of shoots were recorded on days 20 and 30, from
the start of the culture period. Rooting data were obtained on
day 10 following root induction treatments on medium with
IBA. Altogether, ten explants were used in each of two
replicates for each treatment, and the experiment was repeated twice. All data were statistically analyzed by
ANOVA followed by Duncans multiple range test for mean
comparison.
A. tumefaciens-mediated transformation studies. Leaf
explants from axenic cultures were used for transformation
studies. Media composition for co-cultivation [MSH+AS
MS medium with phytohormones 17.80 M BAP, 2.28 M
IAA, and 200 M Acetosyringone (AS)], selective shoot
induction and elongation (SSIEMMS medium with phytohormones 17.80 M BAP+2.28 M IAA+20 mg/l hygromycin+200 mg/l Ceftoxamine), as well as selective shoot
rooting medium (SSRMMS medium with 4.9 M IBA+
25 mg/l hygromycin) are listed in Table 2. The antibiotics and
AS were filter-sterilized and added to autoclaved medium
when the temperature cooled to 40C. The number of
responding explants, shoot buds per explant, and number
and length of elongated shoots were recorded using a stereomicroscope (Nikon, Japan) at 20 and 30 d following
incubation.

155

Bacterial culture, co-cultivation, and hygromycin sensitivity.

Transformation studies were conducted using A. tumefaciens
strain LBA4404 (van der Fits et al. 2000) harboring the binary
vector pCAMBIA1301 (CAMBIA, Australia). The T-DNA of
pCAMBIA1301 contains the intron-interrupted -glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes
under control of the cauliflower mosaic virus 35S promoter. A
50-ml Agrobacterium culture was agitated overnight in liquid
YEB medium (pH 7.2, with rifampicin 25 mg/l, kanamycin
50 mg/l, and chloramphenicol 75 mg/l) at 28C. Bacterial
cells were harvested by centrifugation at 3,000g for
10 min, and the pellet was resuspended to an OD600 of 0.6
with liquid MS basal medium containing 200 M AS. Leaf
explants derived from axenic leaf cultures of 1 cm square were
inoculated in the above bacterial suspension for 3045 min,
with gentle shaking. After excess bacterial suspension had
been blotted dry with sterile filter paper, leaf explants were
co-cultivated on MSH+AS (MS+17.80 M BAP+2.28 M
IAA) in the dark for 0, 1, 2, 3, or 4 d at 252C. To determine
the selection pressure of hygromycin, the untransformed leaf
segments were cultured on SSIEM with different concentrations of hygromycin (0, 5, 10, 15, 20, 25, or 30 mg/l).
Each 9-cm Petri dish was cultured with 25 explants for transformation studies.
Regeneration, subculturing, and rooting of transgenic
plants. In order to prevent bacterial overgrowth, the cocultured explants were initially washed with sterile distilled
water followed by liquid MS basal medium containing
200 mg/l Cefotaxime. The above explants were then cultured on SSIEM supplemented with 17.80 M BAP,
2.28 M IAA, 25 mg/l hygromycin, and 200 mg/l Cefotaxime. Hygromycin-resistant cultures were transferred to fresh
SSIEM at 10-d intervals. Elongated shoots were transferred
to root induction medium containing 4.9 M IBA and
25 mg/l hygromycin. In vitro rooted plantlets were gently
washed under running tap water to remove adhering medium and transferred to plastic pots containing a mixture of
vermiculite and perlite (1:1). The potted plants were hardened in greenhouse as described above.
GUS histochemical assays. GUS expression in co-cultivated
explants and putatively transgenic plants was histochemically assayed according to Jefferson et al. (1987). Tissues

Table 2. Media used for the transformation study of B. monnieri L.

Stage

Medium composition

Medium abbreviation

Duration (days)

Co-cultivation
Selective Shoot Induction and
Elongation Medium
Selective shoot rooting

MS+17.80 M BAP+2.28 M IAA+200 M AS

MS+17.80 M BAP+2.28 M IAA+20 mg/l
hygromycin+200 mg/l Cefotaxime
MS+4.9 M IBA+25 mg/l hygromycin

MSH+AS
SSIEM

2
3035

SSRM

1012

156

MAHENDER ET AL.

stained with indigogenic dye were scored. After overnight

incubation in the substrate solution at 37C, the stained
tissues were rinsed several times with 75% ethanol to bleach
the chlorophyll and were observed under a dissecting microscope. The transformation efficiency was calculated by
percent of gus-positive, co-cultivated explants showing
shoot regeneration on the selection medium.
Molecular analyses of transformed plants. Total genomic
DNA from randomly selected putative transgenic plants
(hygromycin-resistant plants) and untransformed control plants
was extracted according to Doyle and Doyle (1990). For
the detection of transgenes (uidA and hpt) in the transformed
plants, PCR was conducted using primers (Forward-5CGACGGCCTGTGGGCATTTCA-3 and Reverse-5TGGTCGTGCACCATCAGCAC-3) generating a 900-bp uidA
gene fragment and primers (Forward-5-TAGAAAAAGCCT
GAACTCACCG-3 and Reverse-5-TATTTCTTTGCCCTCG
GACG-3) generating a 1-kb hpt gene fragment. PCR
reactions were carried out in 20-l reaction volumes containing 0.5 units of Ex-Taq polymerase and 1Taq buffer (Takara,

Figure 1. High-frequency shoot organogenesis from B. monnieri L.

leaf explants. (a) Multiple shoot induction from cut ends of leaf
explants on MS medium supplemented with 17.80 M BAP and
2.28 M IAA. (b) Formation of well-developed shoots on the same
MS medium. (c) Elongated shoots developing on MS basal medium.
(d) Rooting of elongated shoots on MS medium supplemented with
4.90 M IBA. (e) Potted plant from the greenhouse.

Dalian, China), 0.2 mM of each dNTP, 0.5 M of each primer,

and 50 ng of template DNA.
For detection of the uidA gene, PCR conditions were set
as: initial denaturation 95C for 5 min, 30 cycles of denaturation at 94C for 40 s, annealing at 58C for 40 s,
extension at 72C for 1 min, and a final extension at 72C
for 10 min. The PCR conditions for hpt gene detection were
set as initial denaturation at 94C for 5 min, 30 cycles of
denaturation at 95C for 40 s, annealing at 56C for 1 min,
extension at 72C for 40 s, and final extension at 72C for
10 min. Amplified PCR products were electrophoresed in
1% agarose gels and detected using ethidium bromide
staining.
To detect the expression of transgenes by real time PCR
(RT-PCR), total RNA was isolated from in vitro regenerated
(60 d) transgenic plant lines according to Xu et al. (2010)
and treated with DNase I (Takara, Dalian, China) to remove
traces of DNA. Total RNA (2 g) was used as template to

Figure 2. Production of transgenic B. monnieri L. via Agrobacteriummediated transformation. (a) Explants induced profuse shoot organogenesis on MSH (MS medium containing 17.80 M BAP+2.28 M
IAA) without hygromycin. (b) No shoot organogenesis was observed
on selection medium supplemented with 25 mg/l hygromycin. (c)
Transient GUS expression of co-cultivated leaf explants. (d) Multiple
shoot induction from the wounded ends of co-cultivated leaf explants
cultured on selective shoot induction and elongation medium (SSIEM).
(e) Emerging shoots expressing GUS. (f) Putative transgenic shoots
showing blue coloration. (g) PCR analysis of GUS and hpt (h) positive
lines. 15 represent independent transgenic lines, W wild type, M
molecular weight marker.

SHOOT ORGANOGENESIS AND PRODUCTION OF TRANSGENIC BACOPA

157

Table 3. Transformation efficiency mediated by A. tumefaciens strain LBA4404 (pCAMBIA1301) in B. monnieri L.

Experimentz

No. of co-cultivated explants

No. of shooting explants

under hygromycin selection

No. of transgenic
plants/explant

No. of transgenic plants

GUS positivey

Transformation
efficiency (%)

50

32

9.60.51

307.20.41

64a

50

36

10.20.43

367.50.62

72b

3
Mean

50
50

38
36

11.30.25
10.360.15

429.40.43
367.90.48

76b
70.6

Means with different letters were significantly different (p<0.05)

z

Plants grown on regeneration medium containing 25 mg/l hygromycin

Plants showing GUS gene expression at the fully mature stage

synthesize first-strand cDNA with oligo (dT)18 (First-strand

cDNA synthesis kit, Invitrogen, India). PCR of the uidA
transcript was carried out according to the conditions described above. The housekeeping gene actin was used as an
internal control to check the expression levels of transgenes.
Stable integration of hpt gene in the host genome was
confirmed by Southern blot hybridization. Aliquots of 20 g
of total DNA (from seven transgenic lines and an untransformed control plant) were digested with EcoRI and analyzed using standard protocol for Southern blot (Sambrook
et al. 1989). Digested DNA was hybridized with DIGlabeled probe specific to 500 bp within the hpt gene region.
Labeling, hybridization, and chemiluminescent detection
were performed according to the manufacturers instructions
(Roche Applied Science).

present study. During selective shoot regeneration, elongation, and rooting stages on different media (Table 2), the
concentration of hygromycin was gradually increased from
20 to 25 mg/l with diminishing usage of Cefotaxime (from
200 to 0 mg/l). After a 2-d co-cultivation on MS medium
supplemented with 200 M AS, infected explants resulted
in a transformation efficiency of 70.6%. The transformation
efficiency was determined by percent of co-cultivated
explants showing shoot regeneration on selection medium
(SSIEM), as shown in Table 3.
Regeneration of transgenic plants. During shoot organogenesis, shoots developed directly from infected leaf segments
without an intervening callus phase (Fig. 2d). All leaf

Results
Shoot organogenesis via leaf explants. Among the different
regeneration media studied, a combination of BAP
(17.80 M) and IAA (2.28 M) resulted in the maximum
generation of shoots 85.2 2.43, with an average shoot
length of 2.80.22 cm (Fig. 1ac; Table 1). Elongated
shoots, 45 cm in length, showed an efficiency of 90%
rooting on medium containing 4.9 M IBA. Fully developed roots were obtained from cut ends of the elongated
shoots within a 1012-d culture period on the above medium (Fig. 1d). The potted plants exhibited emergence of fresh
leaves within 2 wk of transfer (Fig. 1e) and showed an 85%
survival rate in the greenhouse.
Optimization of hygromycin for selection and co-cultivation
period. Sensitivity of explants to hygromycin was studied
by culturing uninfected leaf segments on MS medium supplemented with 0 to 30 mg/l hygromycin (Fig. 2a). From the
different hygromycin concentrations tested, a concentration
of 25 mg/l and above completely inhibited shoot organogenesis (Fig. 2b). As a result, 25 mg/l of hygromycin was
utilized for the final selection of transformed plants in the

Figure 3. Molecular analysis of transgenic B. monnieri L. (A) RTPCR analysis of GUS and actin gene expression. (B) Southern blot
hybridization for stable integration of hpt gene. WT wild type, M
molecular weight marker, 17 independent transgenic lines.

158

MAHENDER ET AL.

cultures with shoot buds that responded to SSIEM medium

exhibited dark blue GUS staining (Fig. 2e), as did the
elongated shoots (Fig. 2f), demonstrating their transgenic
nature. After a period of 30 d on the same SSIEM medium,
the shoot primordia had elongated adequately for root induction. The elongated hygromycin-resistant shoots were
finally transferred onto rooting medium (SSRM) containing
25 mg/l hygromycin. During this culture period, only
putatively transformed shoots survived the root induction
phase, and non-transformed escape plants were eliminated.
The transformation efficiency was calculated by percent
of co-cultivated explants showing shoot regeneration on
selection medium (SSIEM). Based on the percent of cocultivated leaf explants producing shoots on hygromycin
medium, the average transformation efficiency of 70.6% was
achieved (Table 3). A mean number of 10.40.15 transgenic
plantlets per explant was recorded. After 1012 d on SSRM
medium, the transgenic plantlets were ready for transfer to the
greenhouse. Potted plantlets survived at 80% success rate in
the greenhouse.
Confirmation of transgenic B. monnieri plants. Leaves
from hygromycin-resistant plants showed typical dark blue
after staining for GUS activity, while leaves from untransformed plants were void of GUS activity. Following PCR
analysis, fragments corresponding to 900 bp of the uidA
gene (Fig. 2g) and 1 kb of the hpt gene (Fig. 2h) were
amplified from genomic DNA of all GUS-positive lines,
whereas corresponding bands were not detected in the untransformed control plant (Fig. 2gh). Real time PCR
analysis of PCR positive lines also revealed the amplification of a 900-bp uidA gene fragment (Fig. 3A) from total
RNA. Stable integration of the hpt gene into the B. monnieri
genome was further confirmed by Southern blot analysis of
transformed plants. From the seven transgenic lines tested,
three lines (Fig. 3B lanes 4, 6, 7) showed single integration
bands, and the other lines (Fig. 3B lanes 1, 2, 3, 5) showed
multiple integration patterns. No probe hybridization was
observed in the untransformed control plants.

Discussion
To date, there are two reports of successful A. tumefaciensmediated transformation of B. monnieri (Nisha et al. 2003;
Ramesh et al. 2011). Nisha et al. (2003) described selection
of putative transformed plants produced indirectly via callus
tissue. Ramesh et al. (2011) reported Agrobacterium-mediated transformation using nodal explants. Neither report
showed stable transgene integration in the B. monnieri genome by Southern blot hybridization. However, the transformation efficiencies claimed by Nisha et al. (2003) were
60% and 68.8% by Ramesh et al. (2011), whereas in our

protocol, we have achieved stable transformation efficiency

of 70.6%, supported by DNA blot analysis. We have followed a similar means (percent of co-cultivated explants
showing shoot regeneration on selection medium) to calculate transformation efficiency as published by Mahender et
al. (2011).
The Agrobacterium-mediated transformation system
remains favored by many researchers as it does not involve
sophisticated equipment and produces more frequently
clean transgenic integration events (intact integration and
single copy; Gelvin 2003). The high transformation efficiency
observed in B. monnieri may be related to the efficient regeneration response, as noted for model systems like tobacco and
Arabidopsis. In conjunction, it can be also attributed to the
formulation of optimal plant growth regulators (17.80 M
BAP+2.28 M IAA) for leaf shoot organogenesis, which
has enabled the generation of this highly efficient transgenic
system for B. monnieri. The present report describes a simple,
efficient, and reproducible A. tumefaciens-mediated gene delivery system for B. monnieri. The optimized transformation
protocol will also facilitate studies to produce transgenic B.
monnieri with higher content of pharmaceutical compounds
or modified secondary metabolic profiles in the future by
using specific metabolic engineering.
Acknowledgments The authors are thankful to Prof. V. S. Raju,
Plant Systematics Laboratory, Department of Botany, Kakatiya University, Warangal, India for the help with identification of plant
material.

References
Bhattacharya S. K.; Ghosal S. Anxiolytic activity of a standardized
extracts of Bacopa monnieri: An experimental study. Phytomedicine 5: 7782; 1998.
Dahanukar S. A.; Thatte U. M. Current status of Ayurveda in phytomedicine. Phytomedicine 4: 359368; 1997.
Dar A.; Channa S. Relaxant effect of ethanol extract of Bacopa
monnieri on trachea, pulmonary artery and aorta form rabbit and
guinea pig. Phytother Res 11: 323325; 1997.
Dixon R. A. Engineering of plant natural product pathways. Curr Opin
Plant Biol 8: 329336; 2005.
Doyle J. J.; Doyle J. L. Isolation of plant DNA from fresh tissue. Focus
12: 1315; 1990.
Elangovan V.; Govindasamy S.; Ramamoorthy N.; Balasubramanian
K. In vitro studies on the anticancer activity of Bacopa monnieri.
Fitoterapia 66: 211215; 1995.
Gelvin S. B. Agrobacterium-mediated plant transformation: the biology behind the gene-jockeying tool. Microbiol Mol Biol Rev 67:
1637; 2003.
Goossens A.; Rischer H. Implementation of functional genomics for
gene discovery in alkaloid producing plants. Phytochem Rev 6:
3549; 2007.
Grotewold E. Transcription factors for predictive plant metabolic engineering: are we there yet? Curr Opin Biotechnol 19: 138144;
2008.

SHOOT ORGANOGENESIS AND PRODUCTION OF TRANSGENIC BACOPA

Jain P.; Khanana N. K.; Trehan N.; Pendse V. K.; Godhwan J. L. Antiinflammatory effects of an Ayurvedic preparation, Brahmi
Rasayana, in rodents. Indian J Exp Biol 32: 633636; 1994.
Jefferson R. A.; Kavanagh T. A.; Bevan M. W. Gus fusions: glucuronidase as a sensitive and versatile gene fusion marker in
higher plants. EMBO J 6: 39013907; 1987.
Khan M. Y.; Aliabbas S.; Kumar V.; Rajkumar S. Recent advances in
medicinal plant biotechnology. Indian J Biotech 8: 922; 2009.
Liu X.; Yang C.; Chen M.; Li M.; Liao Z.; Tang K. Promoting
scopolamine accumulation in transgenic plants of Atropa belladonna generated from hairy roots with over expression of pmt and
h6h gene. J. Medicinal Plants Res 4: 17081713; 2010.
Mahender A.; Sadanandam A.; Zhang P. Highly efficient production of
transgenic Scoparia dulcis L. mediated by Agrobacterium tumefaciens: plant regeneration via shoot organogenesis. Plant Biotech
Rep 5: 147156; 2011.
Mahmoud S. S.; Croteau R. B. Metabolic engineering of essential oil
yield and composition in mint by altering expression of deoxyxylulose phosphate reductoisomerase and menthofuran synthase.
PNAS 98: 89158920; 2001.
Mukherjee G. D.; Dey C. D. Clinical trial on Brahmi. I. J. Exp. Med.
Sci 10: 511; 1966.
Murashige T.; Skoog F. A revised medium for rapid growth and bioassay
with tobacco tissue cultures. Physiol Plant 15: 473497; 1962.
Nisha K. K.; Seetha K.; Rajmohan K.; Purushothama M. G. Agrobacterium tumefaciens-mediated transformation of Brahmi
[Bacopa monnieri (L.) Wettst.], a popular medicinal herb of India.
Curr Sci 85: 8589; 2003.
Oksman-Caldentey K. M.; Hkkinen S. T.; Rischer H. Chapter 4:
Metabolic Engineering of the Alkaloid Biosynthesis in Plants:
Functional Genomics Approaches. In: Veerporte R.; Alfermann
A. W.; Johnson T. S. (eds) Applications of Plant Metabolic
Engineering. Springer, Netherlands, pp 109124; 2007.
Palazon J.; Navarro-Ocaa A.; Hernandez-Vazquez L.; Hossein Mirjalili
M. H. Application of metabolic engineering to the production of
scopolamine. Molecules 13: 17221742; 2008.
Ramesh M.; Karthikeyan A.; Vijayakumar K.; Largia M. J. V.; Pandian
S. K. Agrobacterium-mediated transformation of pharmaceutically
important Indian medicinal herb Bacopa monnieri (L.). J Med Plant
Res 5: 23162321; 2011.

159

Rastogi S.; Pal R.; Kulshreshtha D. K. Bacoside A3a triterpenoid

saponin from Bacopa monnieri. Phytochemistry 36: 133137;
1994.
Rosatil C.; Aquilanil R.; Dharmapuril S.; Pallaral P.; Pallaral P.; Marusicl
C.; Tavazzal R.; Bouvier F.; Camara B.; Giuliano G. Metabolic
engineering of beta-carotene and lycopene content in tomato fruit.
Plant J 24: 413419; 2000.
Sambrook J.; Fritschi E. F.; Maniatis T. Molecular cloning: Laboratory
Manual. Cold Spring Harbor Laboratory Press, New York; 1989.
Schafer H.; Wink M. Medicinally important secondary metabolites in
recombinant microorganisms or plants: Progress in alkaloid biosynthesis. Biotechnol J 4: 16841703; 2009.
Shrivastava N.; Rajani M. Multiple shoot regeneration and tissue
culture studies on Bacopa monnieri (L.) Penell. Plant Cell Rep
18: 919923; 1999.
Tripathi Y. B.; Chaurasia S.; Tripathi E.; Upadhaya A.; Dubey G. P.
Bacopa monnieri Linn as an antioxidant: mechanism of action.
Indian J Exp Biol 34: 521526; 1996.
Vaibhav T.; Kavindra N. T.; Brahma D. S. Comparative studies of
cytokinins on in vitro propagation of Bacopa monnieri. Plant Cell
Tissue Organ Cult 66: 916; 2001.
van der Fits L.; Deakin E. A.; Hoge J. H. C.; Memelink J. The ternary
transformation system: constitutive virG on a compatible plasmid
dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol 43: 495502; 2000.
Vohora S. B.; Khanna T.; Athar M.; Ahmad B. Anagelesic activity of
bacosine, a new triterpene isolated form Bacopa monnieri.
Fitoterapia 68: 361365; 1997.
Xu J.; Mahender A.; Sadanandam A.; Zhang P. A reliable and
efficient method for total RNA isolation from various members
of spurge family (Euphorbiaceae). Phyt Chem Analysis 21: 395
398; 2010.
Yang C.; Chen M.; Zeng L.; Zhang L.; Liu X.; Lan X.; Tang K.; Liao
Z. Improvement of tropane alkaloids production in hairy root
cultures of Atropa belladonna by overexpressing pmt and h6h
genes. Plant Omics J 4: 2933; 2011.
Ye X.; Al-Babili S.; Kloti A.; Zhang J.; Lucca P.; Beyer P.; Potrykus I.
Engineering of provitamin A (beta-carotene) biosynthetic pathway into (carotenoid free) rice endosperm. Science 287: 303305;
2000.