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www.wjpr.net Vol 6, Issue 8, 2017.
1325
Kishnaveni et al. World Journal of Pharmaceutical Research
INVITRO MICROPROPAGATION OF BACOPA MONNIERI, ITS
ANTIMICROBIAL ACTIVITY
M.Krishnaveni* and C. Ragina Banu
*Assistant Professor, Department of Biochemistry, School of Bio-Sciences, Periyar
University, Salem-636011.
1M.Phil Student, Department of Biochemistry, School oF Bio-Sciences, Periyar University,
Salem-636011.
ABSTRACT
Bacopa monnieri (BM) is a small, creeping, glabrous, succulent herb
possessing antioxidant, antimicrobial properties and widely used by
ayurvedic system of medicine since olden days. Hence, it was decided
to standardize micropropagation protocol of Bacopa monnieri for
surface sterilization, suitable media for aseptic culture initiation,
establishment and multiplication, rooting, optimization of medium for
callus induction, analyse phytochemical contents, antimicrobial
activity. The best medium observed for initiation and development in
terms of bud breakage percentage, cluster formation and maximum
shoot length were BM1 (MS+0.5 mg/l BAP + 0.5 mg/l NAA) with
shoot length of 4.2 cm; 80% bud breakage and 65% cluster formation
and BM3 (MS+4 mg/l BAP + 0.4 mg/l NAA) with shoot length of 3.8 cm; 100% bud
breakage but cluster formation rate was low but this rate increased further in the
multiplication stage when compared to BM4 (0.5 mg/l BAP + 0.5 mg/l.), BM5 (MS+
1.0mg/l BAP + 1.0mg/l) medium. Multiplication stage was good with BM3 (4 mg/l BAP +
0.4 mg/l NAA) and BM1 (0.5 mg/l BAP + 0.5 mg/l NAA) continued to give best results for
the average shoot length of 5.3 cm in BM3 and 4.60 cm in BM1. Extracts was found to have
antimicrobial activity on K. pneumoniae, P. aerugenosa, B.stearothermophilus, B. substilis
and S. aureus with zones of inhibitions ranging from 10-26mm.
KEYWORDS: Antimicrobial, Brahmi, Callus, Micro-propagation, Medium.
World Journal of Pharmaceutical Research SJIF Impact Factor 7.523
Volume 6, Issue 8, 1325-1344. Research Article ISSN 2277– 7105
*Corresponding Author
Dr. M.Krishnaveni
Assistant Professor,
Department of
Biochemistry, School of
Bio-Sciences, Periyar
University, Salem-636011.
Article Received on
05 June 2017,
Revised on 25 June 2017,
Accepted on 15 July 2017
DOI: 10.20959/wjpr20178-8987
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Kishnaveni et al. World Journal of Pharmaceutical Research
INTRODUCTION
Bacopa monniera commonly known as brahmi belongs to the scrophulariaceae family,
located in the warmer areas of the globe for its revitalizing property. For the maximum
biomass production, it could be harvested between 75-90 days especially during October and
November. It has a very good demand in the market and total usage was one thousand tons
per annum, most of this are obtained by the industry through unorganized harvesting. So, it is
essential to maintain its growth available all the time and efforts were taken to grow via
invitro methods as they reduce time and seasonal fluctuation, also used in traditional
medicine to treat nerve, skin and digestion problems, anxiety, also used as a brain tonic,
antiepileptic, antipyretic, analgesic, antimicrobial, anti-inflammatory agent. Bacopa monniera
was reported for alkaloid brahmine, nicotinine, herpestine, bacosides A.[1]
The present study
was designed with an aim i) to standardize micropropagation protocol of Bacopa monnieri
for surface sterilization, suitable media for aseptic culture initiation, establishment and
multiplication, rooting. ii) to optimize medium for callus induction, maintenance and
regeneration. iii) To analyse phytochemical contents iv) Antimicrobial activity of Bacopa
monnieri callus.
MATERIALS AND METHODS
Collection of plant material
Bacopa planting material was collected from Govt. Medicinal Garden of Gandhi nagar,
Coimbatore. The plants were put under polyhouse/shade area at TIFAC – CORE for
hardening and finally field planted in poplar plantation area under shade. Further healthy
explants were collected from field established plants.
Media used
Murashige and Skoog (1962) Medium was used.
Growth Regulators used
2,4-dichlorophenoxyacetic acid (2,4-D), Indole acetic acid (IAA), IBA(Indole Butryric acid)
alpha-Naphthalene acetic acid (NAA),6-Benzyl amino purine (BAP), Kinetin (Kn).
Explant selection and sterilization
The disease free, young and healthy nodal explants were selected for carrying out study as
young cells are supposed to have retained their totipotency. Standard procedures were
adopted for all the experiments.
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Explant sterilization
The leaves were removed from the explants and then washed under running tap water for 30
minutes in order to wash off the external dust/contaminants. Then the explants were soaked
in an aqueous solution containing 0.2 Bavistin (BASF, India Limited) and 0.03%
Streptomycin for 10 minutes in Laminar flow hood. Gently washed in sterile double distilled
water for 5 minutes for two cycles. Then the explants were immersed in aqueous solutions of
Savlon (Johnson & Johnson) (1.5% v/v chlorohexidine gluconate solution and 3.0% w/v
cetrimide) for 10 minutes and were shaked regularly. Then the explants were washed
thoroughly with sterile double distilled water for 5 minutes (two cycles). After this treatment,
the explants were sterilized with 0.01% Mercuric chloride (Ranbaxy) aqueous solution for 4
minutes. Then the explants were removed from the sterilizing solution and rinsed thoroughly
for two times with sterile double distilled water.
Initiation of Cultures
Sterilized explants were transferred aseptically to sterilized glass plate under the laminar flow
hood. Then a cut was given on both basal as well as the top portion of the explants to remove
undesirable/dead portions after surface sterilization. The forceps were earlier rinsed in the
70% ethanol and were flamed and then kept for sometime to get cool. Then the lid from one
test tube was removed and test tube's mouth was flamed to avoid any chance of
contamination. Each nodal explant was then placed in an erect position in the test tube
containing medium with the help of long forceps. The lid was finally closed carefully, flamed
lightly and sealed with Klin film. The forceps were then again rinsed with 70% alcohol to
avoid any chance of cross contamination. The same procedure was undertaken for all the
explants. These jars were finally kept in the growth room with temperature conditions 25±
2°C, with a photoperiod of 16 hours daylight and 8 hrs night break under the cool white
fluorescent light of average 2500 lux (cool white fluorescent tube light 40 W GE).
Establishment of Cultures
After approximately 9-10 days of inoculation, the axillary bud break was seen in some
explants. When the explants attain bud proliferation, these cultures were then transferred to
jars containing fresh medium. After 21- 25 days of incubation with a clean and sterilized
forceps under the laminar flow hood, the initiated plants were taken out the test tube, medium
adhered to the plants was removed, undesirable/brownish leaves were removed from the
plants and were taken to the culture bottles containing autoclaved semi-solid media having
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Kishnaveni et al. World Journal of Pharmaceutical Research
the same combinations as that for the culture initiation. Then the bottles were placed in the
culture room under the standard conditions of temperature (25± 2°C) for 16/8 hrs of day/night
break under the cool white fluorescent light of average 2500 lux (cool white fluorescent tube
light 40 W GE).
Axillary Shoot Proliferation
Multiplication of shoots by repeated sub-culturing in multiplication media
The preparation and sterilization steps for the medium, instruments and chamber were
repeated as before. Multiple shoots/cluster were transferred from the culture bottle to a sterile
glass plate using flamed sterilized forceps, the brown leaves were removed from the primary
shoots and sectioned into one node piece after removing the leaves. These nodal segments
were transferred to the multiplication media. All this work was done with extreme care and
inside the laminar flow hood to avoid any possible chance of contamination. These culture
bottles were then incubated in the growth room. These steps were repeated every 25-30 days
for the next sub-culturing. The medium code and medium details are given below:
Medium Code Medium details
MS MS basal as control
BM MS + 0.1 mg/l BAP + 0.1 mg/l NAA
BM1 MS + 0.5 mg/l BAP + 0.5 mg/l NAA
BM2 MS + 1.0 mg/l BAP + 0.2 mg/l NAA
BM3 MS + 4.0 mg/l BAP + 0.4 mg/l NAA
BM4 MS + 0.5 mg/l BAP + 0.5 mg/l Kn
BM5 MS + 1.0 mg/l BAP + 1.0 mg/l Kn
Rooting of the Shoots
Axillary shoots developed in cultures in the presence of cytokinin generally lack roots. To
obtain full plants the shoots must be transferred to a rooting medium, which is different from
the shoot multiplication medium, especially in its hormonal composition. A low salt medium
is found satisfactory for rooting of shoots in large number of plant species.
Rooting Protocol
In the laminar flow, under sterile conditions the Klin film and cap was removed from the
culture bottles in sterile condition (laminar flow hood) and with the help of sterile forceps the
multiplied shoots were removed from the medium and placed on the sterile glass plate. With
the help of sterile scalpel elongated shoots up to 1-2 cm in length were cut and placed into the
rooting medium. The culture bottles were then capped and placed in the growth room under
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the same condition. The time required for in vitro rooting of shoots may vary from 10-15
days. Different combinations of the rooting media used are given below:
Different Combinations of the Rooting Media
MS MS + Sucrose (30 gm/l)+ Agar (8gm/l)
RT1 MS + Sucrose (0 gm/l)+ Agar (7gm/l)
RT2 MS + Sucrose (0 gm/l)+ Agar (8gm/l)
RT3 MS + Sucrose (10 gm/l)+ Agar (7gm/l)
RT4 MS + Sucrose (10 gm/l)+ Agar (8gm/l)
RT5 MS + Sucrose (20 gm/l)+ Agar (7gm/l)
RT6 MS + Sucrose (20 gm/l)+ Agar (8gm/l)
RT7 MS + Sucrose (30 gm/l)+ Agar (7gm/l)
Ex vitro rooting
This technique can be used for many plants. The technique is particularly suitable for species
which root easily and it has been said to have special advantages for woody plants in which
secondary thickening is important for proper root function. In many of these plants, the
absence of a vascular cambium may mean that roots formed invitro are incapable of proper
water movement. Plantlets were taken out of the culture bottles (multiplication subculture)
with the help of forceps and washed thoroughly with water to remove any remaining of the
medium. 0.1% Bavistin treatment was given to the plants in order to protect them from the
fungal attack in the near future. Plantlets were separated into single shoots by cutting their
bases gently with the help of blade. Single shoots were dipped in IBA solution (200ppm)
before planting into a hardening mixture. After this the single shoots are carefully planted in
the trays containing soil and agropeat (M/s Varsha Enterprises, Bangalore, India) mixture in
1:1 ratio.
Callus Induction
Callus is a mass of unorganized cells resulting either as a consequence of wounding in plants
or in tissue culture. Callus is either homogeneous, parenchymatous mass or trachery elements
or sieve elements or submerged cells or trichomes. Callus formation has been found in
angiosperms, gymnosperms, pteridophytes and bryophytes. Callus is somewhat an abnormal
tissue, which has the potentiality to produce normal roots and embryoids and in turn develops
into plantlets. Under the stimulus of endogenous growth substances or hormones added to the
medium, the metabolism of cells, which were in quiescent state, is changed and they began
active division. During this period, cell differentiation, which may have been occurring in the
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intact plant, is reversed and this give rise to new tissue, which is composed of meristmatic
and unspecialized cell types.
Although callus remains unorganized, as growth proceeds, some kinds of specialized cells
may be again formed and which can give rise to organs such as roots, shoots and embroys.
For plant cells to develop into a callus it is essential that the nutrient medium contain plant
hormones, i.e. an auxin, a cytokinin and gibberellins. The absolute amounts of these, which
are required, vary for different tissue explants from different parts of the same plant and for
the same explant from different genera of plants. Callus tissue originating from herbaceous
species material regenerates much better than material from woody plants. When subculture
regularly on nutrient medium callus culture will exhibit a s-shaped or sigmoidal pattern of
growth, which consists of lag, exponential, linear, deceleration and stationary phase. Callus
growth can be monitored by fresh weight and dry weight measurements but dry weight
measurements are more accurate but this requires sacrifice of the sample.
Procedure
Three types of explants: Leaf segments; internodal segments and nodal segments were used
as a source of explant. Explant used for callus induction were taken from established cultures
of Bacopa. The medium employed was MS Basal with different concentration and
combinations of phytohormones such as NAA, Kinetin and 2,4-D. After inoculation the
culture bottles were properly capped and sealed. After labeling, these are transferred to the
incubation room where they are incubated at 25±2°C in the rack covered with black paper.
Different combinations of the callus medium used are given below:
Different Combinations of the Callus Medium
Medium Code Medium details
MS MS basal as control
BCM MS basal as control
BCM1 MS + 0.5 mg/l BAP + 1mg/l 2,4-D
BCM2 MS + 0.5 mg/l 2,4-D
BCM3 MS +1.0 mg/l 2,4-D
BCM4 MS + 2.0 mg/l 2,4-D
Phytochemical screening for callus
Chemical tests were carried out on the callus tissue from micropropagated bottles cut and
grinded well and analysed.
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Alkaloids: About 0.2 g of the extracts was warned with 2% H S0 for two minutes. It was
filtered and few drops of Dragencloffs reagent were added. Orange red precipitate indicates
the presence of alkaloids.
Tannins: Small quantity of extract was mixed with water and heated on water bath. The
mixture was filtered and ferric chloride was added to the filtrate. A dark green solution
indicates the presence of tannins.
Anthraquinones: About 0.5 g of the extracts was boiled with 10% HCl for few minutes in a
water bath. It was filtered and allowed to cool. Equal volume of CHCl was added to the
filtrate. Few drops of 10% NH were added to the mixture and heat. Formation of rose-pink
colour indicates the presence of anthraquinones.
Glycosides: The extract was hydrolyzed with HCl solution and neutralized with NaOH
solution. A few drops of Fehling’s solution A and B were added. Red precipitate indicates the
presence of glycosides.
Reducing sugars: The extracts was shaken with distilled water and filtered. The filtrate was
boiled with drops of Fehling’s solution A and B for minutes. An orange red precipitate
indicates the presence of reducing sugars.
Saponins: About 0.2g of the extract was shaken with 5ml of distilled water and then heated
to boil. Frothing (appearance of creamy miss of small bubbles) shows the presence of
saponins.
Flavonoids: Extract of about 0.2g was dissolved in diluted NaOH and HCl was added. A
yellow solution that turns colourless, indicates the presence of flavonoids.
Phlobatanins: The extract (0.5g) was dissolved in distilled water and filtered. The filtrate
was boiled with 2% HCl solution. Red precipitate shows the presence of phlobatanins.
Steroids: 2 ml of acetic anhydride was added to 0.5 g of the extract of each with 2 ml of
H2S04. The color changed from violet to blue or green in some samples indicating the
presence of steroids.
Terpenoids (Salkowski test): 0.2g of the extract of the whole plant sample was mixed with
2ml of chloroform (CHCl) and concentrated H2S04 (3ml) was carefully added to form a layer.
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A reddish brown coloration of the interface was formed to indicate positive results for the
presence of terpenoids.
Extraction
The whole callus was thoroughly washed. Each parts cut into pieces, dried in an oven at 60°C
for 9hr and pulverized together. The sample was extracted at a solute-solvent ratio of 1:10 for
6h in a soxhlet extractor. The crude extracts were kept in a sample bottles and stored.
Micro-organisms collection and maintenance
The microorganisms used in the study are: Streptococcus faecalis, Bacillus authracis,
Bacillus cereus, Pseudomonas aerugenosa, Escherichia coli, Pseudomonas fluorescence,
Staphylococcus aureus, Clostridium sporogenes, Klebsiella pneumoniae, Bacillus polymyxa,
Ballcillus subtilis and Bacillus stearothermophilus.
Antimicrobial sensitivity testing of the extract against selected bacterial isolates
Susceptibility test were carried out. The medium employed was diagnostic sensitivity agar.
The cultures were prepared in triplicates and incubated at 37°C for 24 to 72 h. 0.2ml of the
broth culture of the test organism was put in a sterile petri-dish and 18ml of the sterile molten
diagnostic sensitivity agar, was added. Wells were bored into the medium using 0.1ml of the
extracts. Streptomycin was used as the standard antimicrobial agent at a concentration of 1
mg ml. The plates were kept in sterilized inoculation chambers for 2 h to facilitate diffusion
of the antimicrobial agents into the medium. The plates were then incubated at 37°C for 24h
and the diameters of the zones of inhibition of microbial growth were measured in the plates
in millimeters.
Thin layer chromatography
Ethyl acetate fractions (10-20ml) were plated on TLC plates (Silica gel 0.25mm) and
developed in benzene: n-butanol: acetic acid (70:25:5). Spots with Rf values identical to
authentic components were identified under UV light (254 nm) by spraying the plates with
Elhmann’s reagent.
RESULTS AND DISCUSSION
Present study on micropopagation of Bacopa was based on research studies.[2-4]
To initiate
the study, nodal explants were taken from field-established plants. The sterilization procedure
includes soaking of explants in an aqueous solution containing 0.2% (Bavistin India Limited)
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Kishnaveni et al. World Journal of Pharmaceutical Research
and 0.03% Streptomycin followed by treatment with 0.01% Mercuric chloride (Ranbaxy)
aqueous solution with an intermediate steps of sterile water wash. Sterilization treatment of
Bacopa, which includes the use of 0.1% Mercuric chloride (w/v) for 2 minutes followed by
rinsing thoroughly with sterile distilled water.[5]
Reports show different sterilization treatment
in which leaves and stem explants were shaken for 10 minutes in Tween-20 and Savlon
(0.3%) v/v chlorohexidine gluconate and 0.6%w/v cetrimide) in water for 10 minutes, rinsed
in running water for 30 minutes, treated with 0.1% Mercuric chloride for 3-4 minutes and
washed several times with sterile water.[2]
However, it was also found that duration of
treatment of mercuric chloride is very critical due to soft and herbaceous nature of explants.
During surface sterilization treatment it was found that treatment with 0.1% Mercuric
chloride,[2,5]
leads to blackening of the explants. Hence, limited treatment of 0.01% Mercuric
chloride was given to the plants. The surface sterilization procedure was optimized and this
helped in preventing blackening of tissues and establishment of clean cultures. The
sterilization procedure initially followed does not include any step with antibacterial
treatment and large number of explants were found contaminated.
Plate.1 Aseptic Culture Initiation (MS Medium)
Plate. 2 Aseptic Culture Initiation (BM1 Medium)
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Plate. 3 Aseptic Culture Initiation (BM3 Medium)
Contamination was controlled after the addition of antibiotic, 0.03% Streptomycin and
antifungal agent, 0.2% and there was no adverse effect on bud sprouting and shoot
multiplication.
Aseptic culture Establishment
Shoot initiation and establishment from Bacopa monnieri nodal explants cultured on MS
basal and MS medium supplemented with various combinations of growth regulators i.e.
BAP in combination with NAA and Kn is described in Table 1. Most of the other research
studies for other medicinal plant species have shown the use of cytokinin alone or in
combination with other in different concentrations. For example for Paederia foetida and
Centella asiatica multiple shoots were obtained in MS medium supplemented with BAP 1.0
mg /litre.[6]
and in Rauwolfia serpentina on MS medium supplemented with benzyladenine
and NAA,[7]
whereas for Bacopa, optimum shoot proliferation was achieved in different
combination of hormones in different concentrations.
During initial week after inoculation, bud initiation was very low. However, bud initiation
was found to be started in most of the cultures from 9-10 days by showing a small newly
sprouted bud, which proliferate into shoot buds with leaves during 21-25 days which were
placed in the culture room under the standard conditions of temperature (25 ± 2°C). All the
experiments were performed thrice with 3 replicates per treatment. Shoot bud initiation was
observed visually on the ninth day of incubation in all replicates in the media having different
concentrations of BAP and Kn.[5]
After 3-4 weeks thick mat of shoot buds spread over 90-
100% of the surface of explant in the presence of 2mM BAP and 4mM Kn. The results of
asceptic culture conditions are shown in Plate 1 to 3.
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Of the four cytokinins (6-enzyladenine, thidiazuron, kinetin and 2-isopentenyladenine),
thidiazurion (6.8 µM) and 6-benzaldehydenine (8.9 µM) proved superior to other treatments,
optimum adventitious shoots buds induction occurred at 6.8 µM.[8]
Plate. 4 Axillary Bud Induction (25 days) in MS Medium
Plate. 5 Axillary Bud Induction (25 days) in BM1 Medium
Plate.6 Axillary Bud Induction (25 days) in BM3 Medium
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thidiazuron(TDZ). However, in our case, we have not used TDZ/2-ip, to ensure that the
protocol standardized be cost effective.
TABLE 1: Observation for axiliary bud induction at 25 days after initiation
Medium Contamination Bud breakage Avg. shoot
length (cm)
Cluster
formation
MS 0.27 ± 0.15 1.00 ± 0.00 2.70 ± 0.17 No cluster
BM1 0.22 ± 0.04 0.80 ± 0.07 4.20 ± 0.32 0.65 ± 0.04
BM2 0.16 ± 0.05 1.00 ± 0.00 3.80 ± 0.12 No cluster
BM3 0.25 ± 0.01 0.68 ± 0.12 3.04 ± 0.54 0.38 ± 0.05
BM4 0.23 ± 0.06 0.77 ± 0.24 3.04 ± 0.54 0.45 ± 0.12
The results of axillary bud induction was shown in plate 4 to 6. It was observed that although
bud break occurred in all the medium under study, following mediums were compared in
terms of bud breakage percentage, average shoot length and percentage cluster formation.
BM3 (0.5 mg/l BAP + 0.5 mg/l NAA) was found to be the best initiation medium in terms of
bud breakage and lower contamination percentage, however cluster formation was observed
to be very low in this media in the initiation stage but was increased after establishment stage
and had became highest during later stages. BM1 (4 mg/l BAP + 0.4 mg/l NAA) was
observed to be having highest shoot length with higher percentage of cluster formation which
was not observed in case of BM3. In the other three mediums: BM4 (0.5 mg/l BAP + 0.5
mg/l Kn) and MS, average shoot length and cluster formation was less as compared to above
defined mediums.
Multiple Shoot Proliferation
After 25-30 days of first subculture, established cultures were transferred to culture jars
having respective media combinations. Multiplication of shoot cultures was carried out by
culturing nodal segments/clusters excised from in vitro raised plants. Maximum numbers of
plants were obtained on medium containing Kin/2-ip (0.1 mg/l) and Kin (1mg/l) in shoot tip
and nodal cultures of Bacopa respectively.[9]
Out of two cytokinins used BAP was found to be more suitable than Kn as BAP resulted in
quicker and better response then the latter while addition of NAA (0.2mg/l) proved
synergistic. [5,10]
Addition of BAP resulted in the increase in number of shoots, mean shoot
length and number of roots/explant.[4]
Direct regeneration of shoots and roots occurred in
nodal explants in Bacopa on MS medium containing NAA (0.1 mg/l) and BAP (0.5 mg/l).[10]
Addition of higher levels (2.0 and 3.0 mg/l) of BAP in the media, helps in differentiation of
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shoots in 63 and 93% cultures from the nodal portion and shoots attained height of 5 cm in
the ninth week in the above medium. Addition of 0.2 mg/l NAA to the above medium caused
shoot regeneration in 95% of the cultures and shoots attained height of 5 cm in the eighth
week. There is also report for use of high concentration of BAP (9- 10mM) for shoot
regeneration.[11,12]
Both these reports clearly supported our observations as well.
Observations were taken for evaluating the growth of explants by taking parameters like
internodal distance (inter-nodal distance was measured form the third node and measured up
to sixth node from the shoot tip), average shoot length and number of nodes (15 shoots
randomly selected per medium). The experiment was carried out in seven mediums having
different concentrations of growth regulators each with 3 replications, only results of best
medium are given. The medium showing best results was BM3 and BM1 with highest
average shoot length of 5.3 and 4.6 respectively with 100% cluster formation. Sub culturing
was carried out after 25-30 days using the same medium combinations as for initiation and
establishment stages. Shoot clusters obtained in each subculture was divided in approx 1cm2
size with approx. 4-5 small shoots in each cluster and inoculated in each bottle.
Final observation was taken after 3rd subculture stage after initiation with 3 replication per
treatment and summarized in the Table 2. Cluster formation initiated from the basal node;
progressively increases in size from each subculture and number of shoots initiated from the
nodal portion vary from medium to medium. Each explant was transformed into a dense mass
of profusely regenerating shoot buds (as“cluster formation” in our case).[5]
It was observed that in case of BM3 highest number of shoots (>3 cm) originated from the
basal node and average number of shoots formed in case of BM3 is 18.8; 10.3 in BM1; 6.7 in
BM4 and lowest no. of shoot regenerated in case of MS is 3.20. In term of number of
nodes/explant, same order is followed i.e. BM3 having 7.3, BM1 having 6.95, 4.3 in BM4
and in case of MS is 3.26. The texture of leaf was succulent and fleshy. It was also observed
that no. of clusters obtained by dividing each mother culture (cluster) ranging from 1.0(MS)
to 4.1-4.4(BM3 and BM1) and hence can be ascribed as suggested multiplication rate for
each medium.
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TABLE 2: Observation of multiple shoot proliferation
Medium
No. of
shoots>3 cm
No. of
nodes
Avg.shoot
length
Internodal
distance
MS 3.20 ± 0.28 3.26 ± 0.00 2.42 ± 0.24 0.72 ± 0.01
BM1 10.3 ± 0.07 6.95 ±0 .00 4.60 ± 0.18 0.79 ± 0.00
BM3 18.8 ± 0.13 7.30 ± 0.09 5.30 ± 0.11 0.85 ± 0.09
BM4 6.70 ± 0.12 5.30 ± 0.07 2.90 ± 0.22 0.78 ± 0.07
It was observed that during each passage, the number of leaves/shoots has increased
substantially along with the height of shoots. The leaf size was approximately 0.4-0.6cm. It
was observed that morphogenic responses exhibited in the form of shoots or roots are
correlative to a specific auxin /cytokinin ratio. In comparison to MS basal media, for BM3,
there is a significant increase in the number of shoots (5.87 times) and average shoot length
(2.19 times). According to previous reports number of shoots per explant was significantly
higher in BA supplemented agarified medium as compared to BA free medium, however
there is a decrease in shoot length with progressive sub culturing in BA supplemented
medium.[4]
Rooting
After two cycles of multiplication subculture, elongated shoots of 2-3 cm in length were
excised and cultured on MS basal medium having different combinations of sugar and agar
with MS basal (MS+ Sugar 30 gm/l +Agar-8 gm/) as control. The experiments were
conducted twice, with 3 replications (with 3 shoots per bottle).
Rooted shoots were taken after 2 weeks, shoot length, root length and no. of roots per explant
(total 9 explants per treatment each time), fresh wt. and dry wt. (keeping them in an oven
with 500°C for 24 hrs) were measured.
Initiation of rooting took place after 5-6 days of inoculation. Single and multiple roots were
formed from the base and the nodal portions and the length of the roots were 1-2 cm within 8-
10 days.
TABLE 3 –Observations for rooting
Medium
Root
length(cm)
Shoot
length(cm)
Shoot/
Root
No. of
roots
Fresh wt.
(gms)
Dry wt.
(gms)
MS 2.12 ± 0.16 3.17± 0.10 2.15 ±.21 5.20 ±0 .04 2.38 ±0 .24 1.47 ± 0.02
RT1 1.50 ± 0.24 2.75± 0.24 1.83 ± 0.70 3.35 ± 0.04 1.90 ± 0.02 1.16 ± 0.04
RT2 2.40 ± 0.01 3.20± 0.09 1.33 ± 0.04 6.00 ± 0.14 1.78 ± 0.01 1.07 ± 0.07
RT3 1.80 ± 0.19 2.60± 0.09 1.44 ± 0.19 9.30 ± 0.14 2.73 ± 0.07 1.79 ±0.02
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Kishnaveni et al. World Journal of Pharmaceutical Research
RT4 2.30 ± 0.09 3.60± 0.24 1.56 ± 0.54 6.10 ± 0.14 2.70 ±.0.06 1.19 ±0.08
RT5 2.02 ± 0.01 6.00± 0.24 2.97 ± 0.04 7.20 ± 0.19 2.54 ± 0.06 1.21 ± 0.08
RT6 2.68 ± 0.09 6.70± 0.09 2.50 ± 0.04 7.72 ± 0.01 2.58 ± 0.06 1.05 ± 0.12
RT7 1.88 ± 0.07 2.88± 0.24 2.50 ± 0.09 5.58 ± 0.04 2.09 ± 0.06 1.18 ± 0.02
The results indicate positive responses. It was observed, that RT5 (MS+agar 7gm/l+sugar
20gm/l) recorded highest S/R (2.97) and 2.6 for RT6 (MS+agar 8 gm/l+ sugar 20gm/l) with
considerable higher no. of roots (7.20 and 7.72 respectively).
Callus Induction
The results of callus induction are shown in Plate 7 and 8. Callus induction requires the
presence of auxins or cytokinins or both in the nutrient media depending on the source of
explant. Callus initiation was carried out by using leaf segment as source of explant. Initiation
was carried out using different growth regulators such as BAP, IAA and 2,4-D containing
media. The explants enlarged with in 12-14 days of inoculation; however callus formation
started after 20-25 days at the ends of the explant. Appearance of callus was globular and was
of pale yellow in color. In the medium BCM (MS +0.5 mg/l BAP + 1mg/l 2,4-D), rapid
callus growth was observed which however turned pale yellow and of globular appearance.
Plate.7 Callus Induction and Proliferation in BCM Medium
Plate.8 Callus Induction and Proliferation in BCM Medium
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Kishnaveni et al. World Journal of Pharmaceutical Research
Reports show, that addition of 1mM BA alongwith 2,4- D, induced a thin layer of granular
callus after 4 weeks of culture. However, all the explants turned brown to black at the base
after 6 weeks of culture.[5]
In case of BCM1 (MS +1 mg/l BAP + 1 mg/l IAA) initial small globular callus was formed,
from where small shoot buds have been regenerated after 20-25 days. From an area of one
cm2 of callus, 3-4 shoots of height 1-1.5 cm were formed. Previous research show, that IAA
induced callus at the cut ends of both stem and leaf explants along with 1-5 shoots per
explant, while in combination with BA, it induced only shoots (1-10 per explant) within 2
weeks after incubation. These shoots attained height of av. 4-5cm after placing in light for 8-
9 days when transferred into multiplication medium i.e. BM (MS +4 mg/l BAP + 0.4 mg/l
NAA). The response shown by regenerated shoots in multiplication medium was similar to
the response shown by explants inoculated in multiplication medium in light. Loose jelly type
callus formation was observed in BCM2 (MS+ 0.5mg/l 2,4-D), BCM3 (MS+ 1mg/l 2,4-D)
and BCM4 (MS+ 2mg/l 2,4-D) which however turned brown after few days. Response of
callus induction varies with the type of explant. However, when callus explants with
regenerated shoot buds were transferred to BCM, further regeneration did not occur and
callus formation/reversion to callus phase started and when the same was transferred to
BCM1 then callus again started showing regeneration threads after 9- 10 days in dark. Callus
formation from nodal explants of Bacopa cultured on MS medium containing 0.5 mg/l 2,4-
dichlorophenoxyacetic acid (2,4-D). Previous reports say, that callus induction was observed
from hypocotyl, root and cotyledonary leaf segments, grown on Murashige and Skoog (MS)
medium supplemented with various concentrations and combinations of 2,4-
dichlorophenoxyacetic acid (2,4-D) and kinetin (Kn).[13]
TABLE 4: Observation for the callus induction medium
Medium Contamination Regeneration Appearance
BCM No No Globular, pale yellow
BCM1 No No Nodular, creamish
BCM2 No No Jelly type brown
BCM3 No No Jelly type brown
BCM4 No No Jelly type brown
Phytochemical screening
The result of the whole callus extracts of Bacopa monnieri showed that ethanol extracts
contain a greater proportion by mass of the component compounds. The medicinal properties
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Kishnaveni et al. World Journal of Pharmaceutical Research
of the plant could be attributed to the presence of one or more of the detected plant natural
chemicals such as alkaloids, tannins, anthraquinones, glycosides, reducing sugars, saponins,
flavonoids, steroids, phlobotanins, terpenoids.
Antimicrobial activity
Callus extracts were positive for saponin and alkaloids; class of compounds that are known to
be effective for the treatment of syphilis and other venereal disease. Hexane and ethyl acetate
extracts were positive for steroids. It should be noted that steroidal compounds are of
importance and interest in pharmacy due to sex hormones. Phytochemical screening of the
extracts revealed the presence of different functional groups. However, the pharmacological
actions of the plant cannot be ascertained by the result of the phytochemical analysis only.
This extracts showed antimicrobial activity against Klebsiella pneumoniae, Clostridium
sporogenes, Pseudomonas aerugenosa, Bacillus stearothermophilus and Bacillus substilis
with zones of inhibition ranging from 10-20mm. Extracts was found to have antimicrobial
activity on K. pneumoniae, P. aerugenosa, B. stearothermophilus, B. substilis and S. aureus
with zones of inhibitions ranging from 10-26mm, revealing its great medicinal potentials for
the treatment of gastroenteritis and pneumonia. The obtained results shows the inhibitory
activity of the extracts (Table.5) and their potential use in the treatment of microbial induced
ailments.
TABLE 5: Antimicrobial sensitivity testing
Microorganism Gram S Extracts Callus (20 mg ml)
Zone of inhibition (mm)
K. pneumoniae(ICIB 418)
C. sporogenes (ICIB 532)
P. fluorescence (ICIB 756)
E. coli(ICIB 86)
P. aerugenosa(ICIB 856)
B. stearothermophihus (ICIB8222)
B. subtilis (ICIB 610)
B. polymysa(ICIB 956)
S. aureus (ICIB 8588)
B. cereus (ICIB 69)
B. mobiles(ICIB 556)
S. aureus (ICIB 155)
S. litmus(ICIB 615)
S. feacalis(ICIB 335)
S. misiles(ICIB 855)
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
22
18
16
13
14
19
24
20
14
17
18
16
13
14
17
23
20
22
21
16
16
14
15
14
11
18
19
17
16
14
24
20
24
20
14
17
17
18
19
17
16
14
15
14
11
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Thin layer chromatography
Figure. 1 showing TLC plates of pink spot derivatives of tissue extracts separated by
thin layer chromatography.
Table 6: Rf values of derivatives of tissue extracts separated by thin layer
chromatography
Plate Rf Value
1 0.81
2 0.74
3 0.83
4 0.84
5 0.75
6 0.68
Fig.1 and Table.6 shows the results of TLC performed for callus tissue extracts and its Rf
value. The observed Rf value was found to be in the range of 0.68 to 0.84.
CONCLUSION
Herbs are being used since ancient time to maintain health, to treat disease and regain the
healthy state of mind and body. They have been used in traditional forms of Indian medicine
and have provided solutions to even those health problems that have defied modern science.
However, due to over exploitation they are on the verge of extinction. In a recent threat
assessment exercise undertaken by TRAFFIC India (Trade Records Analysis of Flora and
Fauna in Commerce) set up by the World Wildlife Fund and the World Conservation Union)
33 plants have been placed on the critically endangered list and 17 on the endangered list.
There are 16 in the vulnerable category and seven that are near threatened. Deforestation and
rapid urbanization have eroded the natural agro climatic spaces in which these herbs grow.
Overuse, unsustainable cultivation practices, illegal export and trade are scrooges that have
further increased the vulnerability of these plants. All these factors put together have made
the ecosystem so vulnerable that they may well destroy India’s 5000-year-old natural health
legacy. Bacopa monnieri L. Penn., commonly known as Brahmi, has been used in Indian
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Kishnaveni et al. World Journal of Pharmaceutical Research
System of Medicine for centuries for everything from snakebite to headache. It is used most
often as a brain tonic and a memory enhancer. The demand of Bacopa is met from natural
population, which leads to put heavy strain on existing natural population and hence slow
depletion of this important herb. Tissue culture techniques can be used to attain rapid
multiplication of the elite clones and germplasm conservation of Bacopa monnieri. In the
present work we have attempted to develop suitable micropropagation protocol and tried to
improve existing protocol of Bacopa monnieri. It was observed that best medium for
initiation and development in terms of bud breakage % age, cluster formation and maximum
shoot length were BM1 (MS+0.5 mg/l BAP + 0.5 mg/l NAA) with shoot length of 4.2 cm;
80% bud breakage and 65% cluster formation and BM3 (MS+4 mg/l BAP + 0.4 mg/l NAA)
with shoot length of 3.8 cm; 100% bud breakage but cluster formation rate was low but this
rate increased further in the multiplication stage. In the other three mediums: BM4 (0.5 mg/l
BAP + 0.5 mg/l.), and MS, shoot length and cluster formation was less as compared to above
defined mediums.
Further in the multiplication stage the trend which we have obtained during the initiation and
establishment phase continued and it was observed that BM3 (4 mg/l BAP + 0.4 mg/l NAA)
and BM1 (0.5 mg/l BAP + 0.5 mg/l NAA) continued to give best results for the average shoot
length of 5.3 cm in BM3 and 4.60 cm in BM1. It was observed that in case of BM3 highest
number of shoots (>3 cm) originated from the basal node and average number of shoots
formed in case of BM3 is 18.8; 10.3 in BM1; 6.7 in BM4 and lowest no of shoot regenerated
in case of MS is 3.20. In term of number of nodes/explant, BM3 having 7.3, BM1 having
6.95, 5.3 in BM4 and in case of MS is 3.26. It was also observed that no of clusters obtained
by dividing each mother culture (cluster) is ranging from 1.0(MS) to 4.1-4.4(BM3 and BM1).
ACKNOWLEDGEMENT
The author would like to acknowledge Honorable Vice- Chancellor and Registrar of Periyar
University, Salem, Tamil Nadu, India.
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