invitro micropropagation of bacopa monnieri, its

www.wjpr.net Vol 6, Issue 8, 2017.

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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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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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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



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)







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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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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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


1340


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



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


1341


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


1343


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.

REFERENCES

1. Ali Esmail, Al Snafi. The pharmacology of Bacopa monniera. A review. International

Journal of Pharma. Sciences and Research. 2013; 4: 154-159.

2. Mathur Shalini, Kumar Sushil. Phytohormone self-sufficiency for regeneration in the leaf

and stem explants of Bacopa monnieri. Journal of Medicinal and Aromatic Plant

Sciences. 1998; 20(4): 1056-1059.


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3. Tiwari V, Singh BD, Tiwari KN. Shoot regeneration and somatic embryogenesis from

different explants of Brahmi [Bacopa monniera (L.) Wettst.]. Plant Cell Reports. 1998;

17(6/7): 538-543.

4. Tiwari KN, Sharma NC, Tiwari V, Singh BD. Micropropagation of Centella asiatica (L.),

a valuable medicinal herb. Plant Cell, Tissue and Organ Culture. 2000; 63(1): 179-185.

5. Shrivastava N, Rajani M. Multiple shoot regeneration and tissue culture studies on

Bacopa monnieri (L.) Pennell. Plant Cell Reports. 1999; 18(11): 919-923.

6. Singh S, Ray BK, Mathew S, Buragohain P, Gogoi J, Gogoi S, Sharma BK and Deka PC.

Micropropagation of a few important medicinal plants. Annals of Biology (Ludhiana).

1999; 15(1): 1-7.

7. Sehrawat AR, Uppal Sanjogta, Punia Anita. In vitro culture and multiplication of

Rauwolfia serpentina - a threatened medicinal plant. Crop Research (Hisar), 2001; 22(1):

68-71.

8. Tiwari V, Tiwari KN, Singh BD. Comparative studies of cytokinins on in vitro

propagation of Bacopa monniera. Plant Cell, Tissue and Organ Culture. 2001; 66(1):

9-16.

9. Tejavathi DH, Sowmya R, Shailaja KS. Micropropagation of Bacopa monnieri using

shoot tip and nodal explant. Journal of Tropical Medicinal Plants. 2001; 2(1): 39-45.

10. Srivastava PS, Ali Gayoor, Iqbal M, Narula Alka and Bharti Nisha. Micropropagation of

Bacopa and effects of heavy metals on growth performance. Role of Plant Tissue culture

in biodiversity conservation and economic development by Nandi SK, Palni LMS, Kumar

A (eds) Gyandra Prakshan, Nainital, India. 2002; 325-344.

11. Yang G, Read PE. In vitro shoot proliferation of 5-leaf aralia explants from field grown

plants and forced dormant stems. Plant Cell, Tissue and Organ Culture. 1997; 47:

289-291.

12. Bhuyan AK, Patnaik S, Chand PK. Micropropagation of curry leaf tree (Murraya koenigii

(L) Spreng.) by axilary proliferation using intact seedling. Plant Cell Reports. 1997; 16:

779-782.

13. Rani G, Virk GS, Nagpal A. Callus induction and plantlet regeneration in Withania

somnifera(L.)Dunal. In vitro Cellular and Development Biology-Plant. 2003; 39(5):

468-474.

invitro micropropagation of bacopa monnieri, its

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