Green page: Research Article - NISCAIRnopr.niscair.res.in/bitstream/123456789/7996/1/NPR 5(1)...
Transcript of Green page: Research Article - NISCAIRnopr.niscair.res.in/bitstream/123456789/7996/1/NPR 5(1)...
Green page: Research Article
IPC code; Int. cJ.7 - AOlH 4/00, AOlH 5/04, AOlH 5/06, A61K 35/78
Keywords: Adenine snlphate, Bacopa monnieri, Callusinduction, Micropropagation, Mnltiple
shoots, Benzyl Amino Purine (BAP), Indole-3-Acetic Acid (lAA), Naphthalene Acetic Acid
(NAA),Kinetin, Nodal explants.
..In vitro multiple shoot and root induction of Brahmi [Bacopa monnieri
(Linn.) Penn.] was achieved from nodal explants. Shoot segments obtained from field grown
plants were surface sterilized and cnltured on Murashige and Skoog's (MS) medium without any
hormones to get regular supply of node explants. In order to find out the suitable conditions for
propagation from nodal explants, MSmedium with different combinations of auxins and cytokinins
were tested. All the treatments were found effective in inducing shoots except those with auxin!
cytokinin alone. The percent response of 92-1 00 was observed in 90% of treatments. The effect
of special cytokinin like adenine sulphate on multiple shoot formation from the nodal explantsof B. monnieri was tested on MSmedium fortified with different hormonal concentrations of
auxin and cytokinin. High percentage of multiple shoot formation (96.3) and maximum numberof shoots (16.33) was observed in the combinational treatmentoflAA (0.2 mg/l) and BAP(1.5
mg/l) . Addition of adenine sulphate at 60mg/l along with lAA(0.2mg 11)and BAP(1. 5mg/l) was
found most effectivein inducing highest number of shoots (18). Allthe other tested combinations
have little effect on increasing the number of shoots. In vitro propagated plants were successfully
transplanted to plastic pots containing sterile soil with 85% survival rate and later establishedunder ex vitro condition.
Bacopa monnieri (Linn.)Penn. (Hindi-Brahm i) is a creeping,
succulent herb belonging to the familyScrophulariaceae. It is one of the few
Ayurvedicherbs with high economic and
scientific values with potential forresearch in tissue culture and
phytochemistry. In India whole plant or
aerial part o~the plant is used to support
healthy blood sugar levels, to prevent the
effect of ageing, to develop intelligence,
to promote mental health and to increase
the body resistance against diseases
(Kapoor, 1990). The principal active
factors that have been reported from this
plant are two steroidal saponins, bacoside
Aand B (Chatterji et ai, 1963). Bacoside
Aand B are optical isomers and bacoside
B may be an artefact, formed during theisolation of bacoside A (Rastogi &
M Ramesh*, R M Saravanakumar and S Karutha PandianDepartment of Biotechnology, Alagappa University,
Karaikudi 630 003, Tamil Nadu, India
*Correspondent author, E-mail: [email protected]; Phone: (04565) 225215
Received 15 March; Accepted 12 September 2005
Mehrotra, 1990). Other bacosides have
been identified as dammarane type
saponins, known as bacosaponins (Garaiet aI, 1996). Other constituents include
alkaloids (herpestine and brahmine),
flavonoids, glycosides, betulic acid and
phytosterols (Jain & Kulshreshtha, 1993).
In addition, Bacopa extract has anxiolytic,
cognition enhancing (Bhattacharya &
Ghosal, 1998), relaxing (Dar & Channa,1997), antioxidant (Mukherjee & Dey,
1996), anticancer (Elangovan et aI,
1995) and immuno-modulatory activities(Dahanukar & Thatte, 1997).
The increase in the number of
people with poor body health has made
the alternative medical practitioners andpharmaceutical companies to overexploit
many plants. Unscrupulous and
unscientific management practices and
ever increasing demand for
phytochemicals have threatened the
existence of most of the medicinal plantsincluding B. monnieri. In vitromicropropagation techniques paved the
wayfor multiple shoot and root induction
in large number of medicinal plants in ashort span of time to meet their demand
in drug industries. Brahmi has been
previouslypropagated using tissue culturetechniques. Thakur et al C1976a)
Abstract
Introduction
m Natural Product Radiance
=============G=--re=--e=--ft==l@!!le:ResearchArticle
Fig. 1: In vitro micropropagation from nodal explants of Bacopa monnieri (A) Shoot tipculture of B. monnieri; (B) Nodal explant developing callus like growth with large number ofshoot buds; (C) Explant showing large number of shoot buds with developed shoots;(D) Micropropagated shoots of 9cm on MS medium with 0.2 mg/l NAA and 1.5mg/l BAP;(E) Effect ofNAA (O.lmgll) and BAP (left panel) and IAA (0. Img/l) and BAP (I.Omg/l) on callusformation; (F) Micropropagated shoots (16.33 per explant) onMS medium with 0.2mg/l IAA and
1.5mg/l BAP; (G) Root initiation from micropropagated plant on MS medium with 0.2mg/lIAA,1.5mg/1 BAP and 60mg/l adenine sulphate after 28 days .in culture; (H) Plantlets
micropropagated in the presence (left) and absence (right) of adenine sulphate.
observed the role of benzyl amino
purine on shoot bud regeneration
from single epidermal cells of
Brahmi. The same group later
reported the development of
abnormal plantlets with alternate
phyllotaxy, instead of opposite
arrangement (Thakur et al, 1976b).
Totipotent ability of distal ends of
B. monnieri internode segments in
regenerating shoots has been
reported on medium without any
hormones (Thakur & Ganapathy,
1978). Morphogenic potential of
node, inter node and stem explants
have been reported successfully(Tiwari et aI, 1998; Shrivastava &
Rajani, 1999; Allet aI, 1999; Tiwari
et aI, 2001). In these reports the
shoots established were again
transferred to full or half strengthmedium with auxin alone for
rooting. Recently, Mohapatra and
Rath (2005) achieved
micropropagation from leaf and
nodal segments and analyzedbiochemical parameters to establish
the sustainabilityofplants. Regardingits genetic improvement, only one
group reported successful protocol
for genetic transformation of this
species using Agrobacteriummediated transformation (Nisha etaI, 2003). The present study
established reliable and reproducible
protocol for rapid one step multiple
shoot and root regeneration from
nodal explants of the herb usingvarious concentrations and
combinations of auxins and
cytokinins.
Vol 5(1) January-February 2006 m
Green @!Be: Research Article
Effect of NAAlIAA and BAP
Results and Discussion
The nodal explantswere cultured
on MS media supplemented with variousconcentrations of NAA(0.1-0.5mgll) or
1AA(0.1-0.5 mg/l) along with the
combination of BAP (1.0-3.0 mgll). The
effectiveness of nodal segments on
in vitro micropropagation was analyzed
after 30 days of culture initiation. Multipleshoot induction (more than two) was
observed on the 12th day of incubation inall concentrations of hormones tested.
Among the different auxin-cytokinin
In medicinal plants like
B. monnieri, reliable and reproducible
propagation from different explants is a
prerequisite for genetic manipulations liketransformation.
Statistical analysis
All the experiments were
repeated thrice (each with 9 explants) and
the response of explants on multiple shoot
induction was recorded after 28 days of
culture initiation. The data pertaining to
mean percentage of culture showing
response, number of shoots per explants
and shoot length were scored and
presented in mean ± standard error.
to Sigma polypropylene boxes (with two
holes at the bottom) containing
autoclaved garden soil. The plants were
covered with polythene bags with fine
holes and kept in a culture room at
26±2°C and 60-65 % relative humidity
for 8-12 days. After 12 days, the polythene
covers were removed and transplanted to
the field for further growth.
Hardening
Micropropagated plantlets
(7 -9cm in height with 8-10 leaves and8-12 roots) with and without adenine
sulphate treatment were gently washed
under running tap water and transferred
Multiple shoot induction and rooting
About 1.5-2cm long nodal
explants were aseptically excised from
4 week-old axenic seedlings and used as
explants for shoot induction on MS
medium supplemented with various
concentrations and combinations of
hormones. Results were recorded at a
regular interval and final data were scoredafter 4 weeks of culture to study the effect
of auxin and cytokinin and in
combination with adenine sulphate to
induce multiple shoots and buds at
26 ± 2°C with a 16hr photoperiod under
cool white fluorescent light. For each
treatment, a total of nine explants were
used in three different experiments. To
study the role of auxins and cytokinins
alone, same number of explants weretransferred to MS basal medium andtreated as control.
were used for multiple shoot induction.
Along with the above mentioned media,
adenine sulphate (20-100 mgll) was used
for enhancing multiple shoot induction.
The pH of all media was adjusted to 5.7
5.75 before adding 0.8% agar gel (Sigma,
USA). Moltened media of about 30m! was
dispensed into polypropylene magenta
vessels (Sigma, USA)and was autoclavedat 15lb and 121°C for 18 minutes.
Autoc1avedmedia were kept at 26± 2°Cbefore use.
Culture medium and conditions for
propagation
Plant materials and maintenance
Materials and Methods
Actively growing and healthyshoot material of B. monnieri were
collected from the field grown plants in
the morning. After removing the leaves,
the shoots were cut into pieces (2-3cm),
each containing a single nodal region.
Explants were thoroughly washed under
running tap water for 20 minutes and with
3% (v/v) laboline for 3min followed by
brief washing with sterile distilled water.
Shoot segments were surface-sterilized,
in 70 % (v/v) ethyl alcohol for 30sec
followedby 0.1% mercuric chloride (wlv)for 3min and then rinsed four times withsterile distilled water. After surface
sterilization explants were finely trimmed
again and placed on to 30ml of basalmedium consisted of MS salts and
vitamins (Murashige & Skoog,1962)
supplemented with 3% (w/v) sucrose and
0.8 % (w/v) agar gel (Duchefa, The
Netherlands) in magenta boxes (Sigma,
USA).All cultures were kept at 26±2°C
temperature and at a relative humidity of
70% under 16hr photoperiod. In vitroestablished shoots served as mother stock.
Nodal segments were separated after
28-30 days of culture initiation for
multiple shoot induction and rooting withdifferent concentrations of auxins and
cytokinins (Fig.1A).
Murashige and Skoog's mediumfortified with different combinations of
auxin NAA (0.1-0.5mgll) and IAA(0.1
0.5 mg1) and q10kininBAP(1.0-3.0J11g1)
m~ --'Natural Product Radiance
============G=,=e=en=palle: Resea,thA,';tle
combinations tried, IAA and BAP
formulations exhibited a better response
than NAAand BAP.In general, number of
shoots multiplied per explant and its
length was slightly more on media with1M and BAP than with NAAand BAP'We
found an increase in the percent response
of explants (96.3-100) showing multiple
shoot regeneration with an increase in the
concentration ofBAP from 1-2.0mgll and
NAAfrom 0.1-0.3mgll. However, a furtherincrease in the concentrations of BAP has
no effect on shoot induction. Between the
two auxins (1M and NAA)tested, 1M (0.1,
0.2, 0.3mgll) with BAP (1.0, 1.5 and 2.0
mg/I) was found to be superior in
increasing the number of shoots (14-16
shoots/explant) than NAA (0.1, 0.2,
0.3mgll) and BAP (1.0, 2.0, 3.0 mgll)
(11-12 shoots/explant). It was observed
that there was no significant variation in
shoot length between the media fortified
with 1M and NAA.Highest average lengthof shoot was 9.43cm on the medium
containing 0.2mgll 1M and 1.5mgll BAP
(Table 1). The percentage of explants
exhibiting multiple shoot formation was
found to be 96-100 in all the combinations
tested. Similar kind of observations were
reported from the leaf, inter node and node
explants of the species (Tiwari et at,
1998). Callus-like growth containing largenumbers of adventitious shoot buds with
some developed shoots were observed at
the cut ends of nodal explants (Fig.1B).
After 28 days of culture in the same
medium, most of the shoots attained a
length of 6-9cm. Shoot buds were foundin all tested concentrations and buds were
visually seen in all three replicates. These
results agree with the earlier study on
Bacopa using BAP/KN (Tiwari et at,1998). In this experiment we observed
induction of large number of shoot buds
after 18-22 days of inoculation. Among
the numerous shoot buds induced around
12-14 shoot buds regenerated into shoots
and attained the height of 6-9cm (Fig. 1C).
However, in the presence of 2.5 and 3.0
mgll BAPalong with 0.4 and 0.5mgllNAA/
IAA, only 5-6 shoot buds regenerated
into shoots with a height of 6-7cm
(Fig. ID). The nodal explants cultured on
medium supplemented with 0.2mgll 1Mand 1. 5mg/l BAP facilitated the
development of maximum number of
shoots (16.33) followed by 0.3mgll 1M
and 2.0 mg/l BAP (15.66). Similarly
medium supplemented with 0.2mgll NAA
and 1.5 mgll BAPregenerated a maximum
of 12.33 shoots per nodal explants with
shoot length of 9.43 (Table 1). There wasenormous number of shoot buds that did
not attain the measurable height, as they
spread throughout the surface of the
explants very closely. In general, the nodal
explants cultured on medium with various
concentrations of auxin and cytokinin in
combination have developed healthy dark
green shoots with medium sized leaves
than medium with auxin/cytokinin alone.
Explants cultured on media fortified withIAA/NAA/BAP alone induced shoots at
lesser number (2-3) at alesserfrequency
(72-92) compared to the media
supplemented with combination
treatments of cytokinin and auxin
(Table 1).
We observed greenish callus with
different concentrations of 1M (0.1-0.5
mgll) andBAP (1.0-3.0mgll).Thequantum
of callus-like growth was not much in the
presence of NAA(0.1-0.5 mgll) and BAP
(1.0-3.0 mgll) than that of 1M (0.1-0.5
mgll) and BAP(1.0-3.0 mgll) (Fig. IE) .
Shrivastava and Rajani (1999) studied
callus response from stem and leaf
explants on MS medium supplemented
with NAA,IAA and 2, 4-D. They observed
all the hormones studied, exhibited a
tendency to regenerate in to shoots or
roots rather than callus. Among the levels
of NAA (0.1-0.5 mgll) and IAA (0.1-0.5
mg/I) along with BAP (1.0-3.0 mg/l)
tested, the shoot-bud induction response
was higher in the media containing 1M
(0.1-0.5mgll) and BAP (1.0-3.0mgll). The
percent response of nodal explants
developing the number of shoot buds was
high with increasing the concentration of
BAP up to 1.5mg/l. Among the media
tested, MS medium supplemented with
0.2mg/l IAA and 1.5mg/l BAP gave
maximum number of shoots (16.33 per
explant) and the shoots reached 6.03cm
within 30 days of culture (Fig. IF) . In
B. monnieri the stimulatory effect of
BAP on high frequency shoot bud
regeneration was earlier observed from
leaf, internode and axillary buds of node
explants (Tiwari et ai, 1998), internodes
(Thakur & Ganapathy, 1978), leaf
explants (Shrivastava & Rajani, 1999).
Tiwari et al (1998) reported the influence
of BAP on generating large number of
shoot buds from leaf explants followed
by node and internode. The same group
(2001) reported the role of BAP,
thidiazuron, kinetin and 2-isopentyl
adenine on shoot bud induction node,
inter node and leaf explants. Mohapatra
and Rath (2005) used BAP alone for
multiple shoot proliferation with 95%
frequency with 9.4 mean numbers of
shoots per node culture. In the present
investigation, it was observed that 1M incombination with BAP was more suitable
than NAAin combination with BAP.Similar
effect ofBAP in e~hancing multiple shoots
Vol 5(1) January-February 2006 m
Green ~e: 'eseGrth Art;tle
from the different explants were reported
in many medicinal and other plant species
like Withania somnifera Dunal (Sen
&Sharma,1991), Ocimum sp. (Pattnaik
&Chand, 1996), Vitex negundo Linn.(Chandramu et aI, 2003), Psidiumguajava Linn. (Loh & Rao, 1989), and
Citrus sp. (Maggon & Singh, 1995).
Effect of adenine sulphate
To study the effect of adenine
sulphate in the presence of auxin and
cytokinin on the induction of multiple
shoots, the nodal explants were cultured
on medium supplemented with adenine
sulphate (20-100mg/l) along withdifferent hormonal concentrations of NAA
(0.1-0.5mg/l), IAA (0.1-0.5 mg/l) with
BAP(1.0-3.0mgll). Additionof 20-40mgll
of adenine sulphate to media with
0.1-0.5mg/1 NAA/IAAalong with 1.0-3.0
mg/l BAP had no significant effect on
percent response of shoot induction and
number of shoots produced per explants.
Only the presence of adenine sulphate at
60 mg/l along with all the concentrations
of NAA/IAAalong with BAP (1.0-3.0 g/l)
enhanced the average number of shoots
per explants (Tables 2 & 3) . Similar effect
of adenine sulphate in enhancing shooting
response to a limited extent was reported
in aromatic endangered medicinal plantHemidesmus indicus R. Br. Addition
of 15mg/1 adenine sulphate to mediurr,
with 1.0 mg/l BAP and 0.5mg/1 NAA
enhanced the average number of shoots
from 4.82 to 5.13 (Misra et aI, 2003).
Among the different concentrations of
adenine sulphate tested, addition of 60
mg/l adenine sulphate with 0.2 mg/l IAA
and 1.5 mg/l BAP induced the highest
number (mean of 18) shoots. On medium
with 0.2mg/l IAAand 1.5mg/l BAP only
16.33 shoots were seen after 30 days of
culture initiation. Similarly medium with
0.2mgll NAAand 1.5mg/l generated meanof 12.33 shoots in contrast to 14 on
medium with 0.2 mg/l IAA,1.5 mg/l BAP
and 60mg/l adenine sulphate. Addition of
80 and 100QIg/1adenine sulphate had no
beneficial effect.The increase in multiple
shoot induction in the present study may
be attributed to the synergistic effect of
adenine sulphate with the combinationsof IAAand BAP.
Rooting
The in vitro grown shoots in the
concentrations tested developed bunch of
short, white root system in the same
medium after 18-22 days. Rootinduction
Table 1: The effect of auxins (IAA & NAA) along with cytokinin (BAP) on multiple shoot induction from
the nodal explants of Bacopa monnieri on MS medium
Auxin! Concentration% of explant responseNo of shoots/Shoot length
Cytokinin
(mgll)for multiple shootexplant(em)
induction (Mean±SE)
(Mean:tSE)(Mean:tSE)
1!*
I 72.6 ± 0.41I2.33 ± 0.41 6.33 ± 0.41I
NAA0.1- 82.6 ± 0.41I2.33 ± 0.41 I5.66 ± 0.41
I
iBAP
1.096.3 ± 0.414.33 ± 0.416.03 ± 0.50!NAA+BAP0.1+1.096.3 ± 0.41!
11.00 ± 0.35 8.16 ± 0.08!
NAA+BAP
f0.2+1.5 96.3 ± 0.4112.33 ± 0.419.43 ± 0.04
NAA+BAP
0.3+2.0100.0±0.076.33 ± 0.417.03 ± 0.04
NAA+ BAP
0.4+2.596.3 ± 0.414.33 ± 0.418.00 ± 0.07
NAA+BAP
0.5+3.096.3 ± 0.412.33 ± 0.416.90 ± 0.07I
I 92.6 ± 0.41 3.43 ± 0.407.56 ± 0.06IAA 0.1 -IlAA + BAP
0.1+1.0I96.3 ± 0.41 14.66 ± 0.419.40 ± 0.07
lAA + BAP
0.2+ 1.596.3 ± 0.4116.33±0.418.03 ± 0.05
IAA+ BAP
0.3+2.092.6 ± 0.4115.66 ± 0.419.56 ± 0.04
IAA+ BAP
0.4+2.596.3 ± 0.418.33 ± 0.417.56 ± 0.06
lAA + BAP
0.5+3.092.6 ± 0.415.66 ± 0.41I7.06 ± .04
Each value represents the mean ± SE of 3 replicates with 25-35 nodal explants.
*MS basal medium devoid of any hormonesm____ Natural Product Radiance
Green !!lle: Researlh Arl;lle
Table 2: The effect of adenine sulphate along with different combinations
of NAAand BAPon multiple shoot formation from nodal explants ofBacopa monnieri
Auxin! Concentration Cone.of adenine % of explant responseCytokinin (mgll) ; sulphate for shoot induction
(mg/l) (Mean:l:SE)
1No.of shoots!jexplant(Mean±SE)
was found to be 100% in all
concentrations tested with an average of
14-21 roots per explants with 16-18 shoots
(Fig. 1G). Average length of root system
was 2cm. Rooting did not occur onmedium without IAAlNAA.
Vol 5(1) January-February 2006
Each value represents the mean ± SE of 3 replicates with 30-35 nodal explants.Values given in bold are control for each treatment
NAA+BAP I 0.1+1.0
NAA+BAP I 0.2+1.5
NAA+BAP, I 0.3+2.0
NAA+BAP 0.4+2.5
NAA+BAP 0.5+3.0
o
20
40
60
80
100
o
20
40
60
80
100
o
20
40
60
80
100
o
20
40
60
80
100
o
20
40
60
80
100
96.3:1:0.41
96.3±0.41
96.3±0.41
100.0±0.35
96.3±0.41
92.6±0.41
96.3:1:0.41
96.3±0.41
96.3±0.41
92.6±0.41
100.0±0.35
92.6±0.41
92.6:1:0.41
92.6±0.41
96.3±0.41
96.3±0.41
100.0±0.35
92.6±0.41
96.3:1:0.41
96.3±0.41
96.3±0.41
92.6±0.41
92.6±0.41
92.6±0.41
96.3:1:0.41
92.6±0.41
96.3±0.41
92.6±0.41
96.6±0.41
92.6±0.41
11.00:1:0.35
12.33±0.41
12.66±0.41
13.00±0.35
12.33±0.41
11.00±0.35
12.33:1:0.41
13.00±0.35
13.66±0.41
14.00±0.35
13.66±0.41
12.33±0.41
6.33:1:0.41
6.66±0.41
7.00±0.35
7.33±0.41
6.66±0.41
6.33±0.41
4.33:1:0.41
4.66±0.41
5.00±0.35
5.33±0.41
5.00±0.35
4.33±0.41
2.33:1:0.41
2.33±0.412.66±0.41
3.00±0.35 .
2.33±0.41
2.33±0.41
Transplanting
In vitro propagated plantlets
transplanted into the polypropylene boxes
with autoclaved garden soil recovered
well after 8-12 days and exhibited 80%
survival. Alltransplanted plants showed a·
similar growth habit to field grown plants
(Fig.IH). Plantlets micropropagated andhardened in the presence and absence of
adenine sulphate did not exhibit any
phenotypic difference.Acclimatizedplants
established in the field condition grew
normally and did not show any variationson growth characteristic features when
compared to the control plants.
Conclusion
The nodal explants asepficallyobtained from in vitro multiplied shoot
segments were used for multiple shootinduction using MS basal media
supplemented withvarious concentrations
and combinations of auxins like IAA,NAAand cytokinins like BAP and Adenine
sulphate. Micropropagation was achieved
with the percent response of 92-100 in
most treatments. Ofthe two auxins tested,we found IAAto be more suitable than
NAAfor highest percentage of multipleshoot induction with maximum numberof shoots. Maximum number of shoots
wereobsetvedonMSmediumsupplemented
with O.2mgllIAAand 1.5mgllBAP (16.33
per node). Thepresence of60mg/1adenine
m
Green l!!!Ie: Researth Art;,'e
Table 3: The effect of adenine sulphate along with different combinations
of IAAand BAPon multiple shoot formation from nodal explants ofBacopa monnieri
Each value represents the mean ±SE of 3 replicates with 30-35 nodal explants
Values given in bold are control for each treatment
m
IAA+BAP 0.1+1.0
Acknowledgements
sulphate along with 0.2 mgll 1M and 1.5
mg /1 BAP enhanced the number of
multiple shoots from 16.33 to 18 per
explant. Micropropagated plantlets
transferred to plastic pots grown to
maturity under ex vitro condition.
Although there are several reports on
multiple shoot regeneration from node
explants of B. monnieri, there is no
report on in vitro multiplied shoots with
not more than 18 shoots per explants and
shoot length up to 9cm after 30 days of
culture period. Another significant aspect
of this work is that all the multiplied
shoots developed roots in the same
medium itself and therefore, no need to
transfer them to separate root regeneration
medium. Since this plant appears to
have high potential in drug industry to
prepare high quality drugs on large
scale, supply of pharmaceutically
important raw :naterial can be ensuredthrough this Kind of rapid one step
micropropagation technique. Further
work is in progress on standardizing the
parameters for Agrobacte riurn mediatedtransformation using node explants as
starting material.
1. Ali G, Srivastava PS and Iqbal M,
Proline accumulation, protein pattern
and photosynthesis in Bacopa monnieri
regenrants grown under Nacl stress,
Bioi Plant, 1999, 42, 89-95.
The authors are thankful to Tamil
Nadu State Council for Science and
Technology(TNSCS&T),Chennai, India for
financial support.
Natural Product Radiance
References
No. of
explant(Mean:l:SE)
5.66:1:0.41
5.66±0.416.o0±0.41
6.33±0.35
5.66±O.41
5.33±0.41
8.33±0.41
8.66±o.41
9.33±O.41
9.66±0.41
9.60±0.40
8.66±0.41
15.66:1:0.41
16.33±O.41
16.33±0.41
17.33±0.41
16.66±0.41
15.66±0.41
16.33:1:0.41
16.66±0.41
17.33±0.41
18.00±0.35
17.66±0.41
16.66±0.41
14.66:1:0.41
14.66±o.41
15.33±O.41
15.66±0.41
14.66±0.41
14.33±0.41
96.3:1:0.41
96.3±0.41
96.3±0.41
92.6±0.41
92.6±0.41
92.6±0.41
92.6:1:0.41
92.6±0.41
96.3±0.41
92.6±0.41
96.6±0.41
92.6±0.41
92.6:1:0.41
92.6±0.41
96.3±0.41
96.3±0.41
100.0±0.35
92.6±0.41
96.3:1:0.41
96.3±0.41
96.3±0.41
92.6±0.41
100.0±0.35
92.6±0.41
96.3:1:0.41
96.3±0.41
96.3±0.41
100.0±0.35
96.3±0.41
92.6±0.41
% of explant responsefor shoot induction
(Mean:l:$E)
o
20
40
60
80
100
o
20
40
60
80
100
o20
40
60
80
100
o
20
40
60
80
100
o
20
40
60
80
100
Cone. of adenine
sulphate(mgll)
0.3+2.0
i0.2+ 1.5,
1
IAA+BAP 0.5+3.0
IAA+BAP 0.4+2.5
IAA+BAP
IAA+BAP
Auxin! Cone.
Cytokinin (mg/l)
Green l!!!!le: Researth Artit'e
2. Anonymous, In: Indian Herbal
Pharmacopoeia, Worli, Mumbai: Indian Drug
ManufacturersAssociation, 1998, Volume 1.
3. Bhattacharya SK and Ghosal S, Anxiolytic
activity of a standardized extract of Bacopa
monnieri - an experimental study,Phytomedicine,1998, 5, 77-82.
4. Chatterji N, Rastogi RP and Dhar ML,
Chemical examination of Bacopamonnieri Westtst. Part 1- Isolation of
chemical constituents, Indian J Chem,1963, 1, 212-215.
5. Chandramu C, Manohar Rao D and
Dashvantha ReddyV,Highfrequency induction
of multiple shoots from nodal explants of
Vitex negundo L.using sodium sulphate, J
Plant Biotechnol, 2003, 5(2), 107-113.
6. Dahanukar SAand Thatte UM,Current Status
of Ayurveda in phytomedicine,
Phytomedicine, 1997, 4, 359-368.
7. Dar Aand Channa S, Relaxant effectof ethanol
extract of Bacopa monnieri on trachea,
pulmonary artery and aorta from rabbit and
guinea pig, Phytother Res, 1997, 11,323-325.
8. Elangovan V,Govindasamy S, RamamoorthyNand Balasubramanian K,In vitro studies
on the anticancer activity of Bacopa
monnieri, Fitoterapia, 1995, 66,211- 215.
9. Garai S, Mahato SB, Ohtani Kand Yamasaki
K,Dammarane-type triterperoid saponins from
Bacopa monnieri, Phytochemistry,1996, 46, 815-820.
10. Jain P and Kulshreshtha DK, Bacoside AI, a
minor saponin from Bacopa monnieri,Phytochemistry, 1993, 33, 449-451.
11. Kapoor ill, Handbook ofAyurvedic Medicinal
plants, Boca Raton, CRCpress, 1990.
12. Loh CS and Rao AN, Clonal propagation of
guava (Psidium guajava 1.) from seedlings
and grafted plants and adventitious shoot
formation in vitro, Sci Hort, 1989, 39,31-39.
13. Maggon R and Singh BD, Promotion of
adventitious bud regeneration by ABA in
combination with BAP in epicotyl and
hypocotyl explants of sweet orange [Citrussinensis (L.)Osbeck], Sci Hort, 1995,63,123-125.
14. Misra N, Misra P, Datta SKand Mehrotra S,
Improvement in clonal propagation of
Hemidesmus indicus R.Br. through
adenine sulphate, J Plant Biotechnol,2003,5(4) ,239-244.
15. Mohapatra HP and Rath SP,In vitro studies
of Bacopa monnieri -An important
medicinal plant with reference to its
biochemical variations, Indian J Exp Bioi,2005, 43, 373-376.
16. Mukherjee GD and Dey CD, Clinical trial on
Brahmi, Indian J Exp Med Sci, 1996,10,5-11.
17. Nisha KK, Seetha K, Rajmohan K and
Purushothama MG, Agrobacteriummediated transformation of Brahmi
[Bacopa monnieri (L.)Wettst.], a popular
medicinal herb of India, Curr Sci, 2003,85,85-89.
18. Pattnaik SK and Chand PK, In vitropropagation- of medicinal herbs
Ocimum americanum L. syn. O.canumSims (hoary basil) and O. sanctum(holy basil), Plant Cell Rep ,1996, 15,846-850.
19. Rastogi RP and Mehrotra BN, Compendium
of Indian Medicinal Plants, Central Drug
Research Institute, Lucknow and Publications
& Information Directorate, NewDelhi, Vol. 1,
1990, Reprint edn. 1993, pp. 53-54.
20. Sen J and Sharma AK,Micropropagation of
Withania somnifera from germinating
seeds and shoot tips, Plant Cell Tiss OrgCult, 1991, 26, 71-73.
21. Shrivastava N and Rajani M, Multiple shoot
regeneration and tissue culture studies on
Bacopa monnieri (1.) Pennell, PlantCell Rep, 1999, 18, 919 - 923.
22. ThakurS,GanapathyPSandJohriBN,1976a,In vitro shoot bud differentiation from
epidermal cells of stem segments in Bacopamonnieri (Linn.) Pennell, Beitr BioiPflanz, 1976, 53, 321-330.
23. Thakur S, Ganapathy PS andJohri BN, 1976b,
Differentiation of abnormal plandets in
Bacopa monnieri, Phytomorphology,1976, 26, 422-424.
24. Thakur S and Ganapathy PS, Morphogenesis
and organ differentiation in Bacopamonnieri stem cultures, Indian J ExpBioi, 1978, 16, 514-516.
25. Tiwari V, Singh BD and Tiwari KN, Shoot
regeneration and somatic embryogenesis from
different explants of Brahmi [Bacopamonnieri (L.) Wettst], Plant Cell Rep,1998, 17, 538-543.
26. Tiwari V, Tiwari KN and Singh BD,
Comparative studies of cytokinins on in vitropropagation of Bacopa monnieri, Plant
Cell Tiss Org Cult, 2001, 66(1), 9-16.
Vol 5(1) January-February 2006 ----------------- ~m