Mahatma Gandhi Mission

College of Agricultural Biotechnology, Aurangabad

(Affiliated to Marathwada Agricultural University, Parbhani)
(ISO 9001-2000 Certified)

O.R.W. of Research Project of

Under-Graduate Student, B. Sc. (Agricultural Biotechnology)
Name of student :-
Registration Number:-

Semester: VIIth Semester-2010-11

Major Subject: Agril. Biotechnology

Major Advisor: Dr. B. N. Chavan

Effect of different concentrations of IAA and IBA on Shoot proliferation and Root
initiation of Bacopa monnieri (Brahmi)

INTRODUCTION:-
Medicinal plants are of great interest to the researchers in the field of biotechnology as
most the drug industries depends, in part, on plants for the production of pharmaceutical
compounds (Chand et.al., 1997). Among the world’s 25 best selling pharmaceutical medicines,
12 are plant derived (O’Neill and Lewis 1993). Bacopa monnieri [L.] Pennell belonging to the
family Scrophulariaceae is an amphibious plant of the tropics and normally found growing on the
banks of rivers and lakes. It is commonly known in India as Bramhi or Jala-bramhi. It is small
creeping, glabrous and succulent herb with thick, soft, ascending branches and sessile, obovate
ablong or spatulate leaves; flowers are whitish blue with purple veins on long pedicles. It has a
great market demand due to its high medicinal value. Moreover, because of the heavy demand
and short supply, it is the most adulterated species in Ayurvedic formulations. So there is need to
mass-propagate the selected clones.
Brahmi is also known as “Medhya rasayanas” in ayurveda as it increases mental clarity &
brain stimulating action (Bhattacharya and Ghosal 1998). The medicinal properties of Bacopa
monnieri responsible for improving memory-related function have been attributed to the
presence of different types of saponins such as Bacosides A, B, C, & D called the “memory
chemicals”(Rastogi et. al., 1994). These compounds are attributed with function like to enhance
the transmission efficiency of nerve impulses, strengthening memory & cognition (Singh. et. al.,
1997). Bacopa also contains variety of medically active substances i.e. stigma sterol, sapogenins
& flavonoids. Other compounds are D-mannitol, betulic acid, beta-sisterol, octacosane, nicotine
& amino acid. It also possesses anti-inflammatory, analgesic, antiptretic, epilepsy, insanity,
anticancer & antioxidant activities (Satyavati et. al., 1976; Jain et. al., 1993; Elangovan et. al.,
1995; Tripathi et. al., 1996). Also used for the treatment of asthma, water retention & blood
clearing. Leaf juice of brahmi is given to children for relief in bronchitis & diarrhoea. In
Pakisthan, the herbal drugs, Brahmi-buti, is used to treat skin diseases, leprosy, epilepsy, eczeme,
asthama, hoarseness of the voice, and diseases of the nervous system (Shakoor et. al., 1994).
More than 90% of plant species used by industry are however collected from the wild
source of which 70% involves unorganized harvesting. This factor poses a serious threat to the
genetic stock and the biodiversity of medicinal plant. Natural regeneration is also hampered by
death of plant at two leaf stage & specific habitat requirement. The submerged shoots of Bacopa
can hardly attain the required growth & multiplication. The role of extinction of medicinally
important plant species is further accelerated by habitat degradation, illegal trade practices, and
0loss of regeneration potential of degraded forests, policies and regulations. Recent reports of
National Medicinal Plant Board (NMPB), Government of India and Technology Information
Forecasting and Assessment Council (TIFAC) has recommended immediate attention to few
medicinal plants, among which Bacopa monnieri prominently features, which makes this plant in
the category of highly endangered plants in India..
According to NMPB annual consumption of Bacopa monnieri during the year 2008 is
2548 MT (Wild/Cultivated) with an annual growth rate of 7% annually. Brahmi gives good
income to the farmer. The average yield is about 300 q/ha fresh wt. and 60 q/ha dry wt. It cost
about Rs. 200000/ha from the fresh wt. while the cost of cultivation is 35000/ha. The rate of dry
Brahmi material is about 20 Rs/kg. Thus, the quality planting material can be available for the
farmers.
So on the view of the wider market demand there is needed to mass propagate this plant
and conserve its wild stock. The present study is focusing on the following objectives.

Objectives:-

 Standardization of suitable media for aseptic culture, initiation, establishment & rapid
multiplication by inducing multiple shooting.

 Conservation of Bacopa monnieri under ex-vivo and in vitro condition.

REVIEW OF LITERATURE:-
The attempts has been made in this chapter to review and classify the work done in past
on this aspect of present investigation by eminent scientists in India and abroad.
Morrel 1960 first time used tissue culture methods for micro-propagation of Orchids,
offer an alternative means of plant vegetative propagation. Clonal propagation through tissue
culture popularly called micro-propagation can be achieved in a short time and space.
Tiwari et. al., 1998, investigated morphogenetic potential of node, internodes and leaf
explants of Brahmi were investigated to develop reliable protocols for shoot regeneration and
somatic embryogenesis.
Murashige and Skoog formulated basal medium for the in vitro propagation of
herbaceous plant species by supplementing it with different combinations of growth regulators.
Tiwari et. al., 2000, used nodal explants for in vitro propagation of Bacopa monnieri
using liquid shake cultures with or without 6-benzyladenine. Also tried rooting on different
media in Bacopa, i.e. MS media with or without hormones and found that rooting was highest
(90%) on full strength MS medium containing 2.46 µM IBA.
Tiwari et. al., 2001, reported use of range of cytokinins for multiple shoot induction for
Bacopa, with node, internodes and leaf explants. Out of the four cytokines (6-benzyladenine,
thidiazuron, kinetin and 2-isopentenyladenine) tested thidiazuron (6.8 M) and 6-benzyladenine
(8.9 M) proved superior to other treatments.
Binita et. al., 2005, reported use of cytokinin BAP (1.1 µM) in combination with auxin
IAA (0.2 µM) gives maximum shoot proliferation (7-8 shoot) from nodal explants.
MATERIALS AND METHOD:-
The details of materials to be used and methods will be adopted for conducting the
present investigation is described in this chapter under appropriate heads.
A. Experimental site:
The experiment will be conducted in plant tissue culture laboratory of MGM College of
Agriculture Biotechnology, Aurangabad.
B. Lab Conditions:
The cultures will be incubated in a culture room at controlled environmental condition
under 16 hrs photoperiod provided by cool white fluorescent tubes.
C. Experimental details:
The study will be conducted in the controlled laboratory conditions. The MS basal
medium will be used to study the effects of different concentrations of IAA and IBA on shoot
proliferation and root initiation of Bacopa monnieri. The MS Basal medium will be
supplemented with BA at 1.1 µM/lit (constant for all the treatments) + IAA and IBA at variable
conc. (IAA 0.00, 0.15, 0.20, 0.25 & IBA 0.10, 0.15, 0.20, and 0.25 µM/lit).

Statistical Design : - Completely Randomized Design (CRD)

1. No. of treatments : - 08
2. Total no. of replication : - 03
3. Treatment details: Concentrations of IAA and IBA

Treatment Concentrations per Liter

T0 Control
T1 IAA 0.15 (µM)
T2 IAA 0.20 (µM)
T3 IAA 0.25 (µM)
T4 IBA 0.10 (µM)
T5 IBA 0.15 (µM)
T6 IBA 0.20 (µM)
T7 IBA 0.25 (µM)
Other experimental details:-
1. Explant: -
Aseptically growing Brahmi plants are used as a source of explants. Nodal segment from
aseptic culture of Bacopa monnieri will be used as explants.
2. Media: -
MS basal medium + BA (1.1 µM/lit) + IAA and IBA (Variable Conc. 0.0, 0.15, 0.20,
0.25 & 0.10, 0.15, 0.20, 0.25 µM/lit) + Sucrose 3% + 0.8 % Agar + pH 5.6 - 5.8.
3. Biometric observations:
Explants will be observed after weekly intervals, in respect to
1. Number of shoots initiated
2. Number of roots initiated
3. Height of ex-plant (Shoots)
4. Dry weight of shoot and root will be observed after the end of experiment.
4. Sampling technique:
Each replication of all eight treatments will be observed for the multiple shoots induction,
root initiation and length of shoot.
5. Analysis of data:
The data obtained on various observations was analyzed by “Analysis of variance”
method (Panse and Sukhatme 1967). The total variance (S2) and degree of freedom (N-1)
will be partitioned into different possible sources. The variance due to the treatments will
be compared with the error variance to find out ‘df’ values and ultimately results were
inferred upon the 5% probability (P=0.05) level of significance. The standard error for
factors based on error variance will be calculated. Whenever, the results will found to be
significant, critical difference (CD) will be calculated for comparison of treatment means
at 5 per cent of significance (CD at 5%).
REFRENCES:-
Annonymous 2008; www.nmpb.nic.in.
Bhattacharya SK & Ghosal S 1998 Anxiolytic activity of Bacopa monniriera- An
experimental study. Phytomed, 5:77-82.
Binita BC, Dave MA & Jasraj YT 2005 Bacopa monnieri: rapid efficient & cost effective
micropropogation. Plant tissue culture & Biotech, 15(2): 167-175.
Chand S, Sahrawat AK and Prakash DVSSR 1997 In vitro culture of Pimpinella anisum
L. (Anise) J. Plant Biochem. Biotech. 6: pp. 1-5.
Elangovan V, Govindasamy S, Ramamoorthy N and Balasubramanian K 1995 In vitro
studies on the anticancer activity of Bacopa monnieri. Fitoterapia 66(3)
: pp. 211-215.
Jain P and Kulshreshtha 1993 Bacoside A1 a minor saponin from Bacopa monniera.
Phytochemistry 33: pp. 449-451.
‘O’ Neill M and Lewis A 1993 Human medicinal agents from plants. In: Kinghorn AD
Balandrin MF, ACS Sysmposium Series 534, Washington, DC. p.48.
Rastogi S, Mehrotra BN & Kulshreshtaha DK 1994 proceeding 4th international congress
of enthnobiology deep publication, New Delhi, p.93.
Sattyavati GV, Raina MK & Sharma M 1976 Indian medicinal plants vol-1 Indian council
of medical research, New Delhi, pp. 20-35.
Shakoor Abdul, Akram Mahmood, Asharaf CM and Siddiqui MR 1994 Pharmagonistic
study and chemical/pharmacological evaluation of Brahmi-buti. Hamdarad
Medicus 37(3): pp. 92-109.
Singh HK & Dhawan BN 1997 neuropsychopharmacological effect of the ayurvedic
nootropic Bacopa monnieri Linn. (Brahmi)Ind pharmacol. 29: pp. 359-365.
Tiwari V, Singh BR & Tiwari KN 1998 shoot regeneration & somatic embryogenesis
from different explants of Brahmi (B. monnieri). Plant Cell Rep. 17: pp. 538-543.
Tiwari V, Tiwari KN and Singh BR 2000 Suitability of Liquid cultures for in vitro 
multiplication of Bacopa monniera L. Wettst. Phytomorphology 50: pp. 337-342. 
Tiwari V, Tiwari KN and Singh BD 2001 Comparative studies of cytokinins on in vitro
propagation of Bacopa monnieri. Plant Cell, Tissue and Organ Culture
66(1):
 pp. 9-16.
Tripathi YB, Chaurasia S, Tripathi E, Upadhyay A and Dubey GP 1996
Bacopa monnieri L. as an antioxidant mechanism of action. Indian Journal of
Experimental Biology 4(6): pp. 523-526.

Dr. B. N. Chavan
(Major Advisor and Chairman)

Members of Advisory Committee:

1. Asst. Prof. A. V. Kharde
      Forwarded in triplicate, to _________________________________________ for approval.
      
Approved/ Not-Approved 
 
Principal