ANTIBIOTIC SCREENING FOR ELIMINATION OF

CONTAMINATION BACTERIA IN BAMBUSA

BALCOOA TISSUE CULTURE
Thesis
Submitted to

Shri Rawatpura Sarkar University

Dhaneli, Raipur (C.G.)
For the degree of
Bachelor of Science
IN
Microbiology
(Under the faculty of Applied science)
By Rajdeep Bose
SRUAB0690
Under the supervision of
Mr. Anup Karmakar (Scientist)
Devleela Life sciences,Pvt. Ltd.
Raipur- 492015(Chhattisgarh)

1
 DECLARATION BY CANDIDATE
I Rajdeep Bose the student of Bachelor of Science in Microbiology, under
Department of Science in Shri Rawatpura Sarkar University, Raipur
Chhattisgarh, declare that the thesis entitled “ANTIBIOTIC SCREENING FOR
ELIMINATION OF CONTAMINATION BACTERIA IN BAMBUSA BALCOOA TISSUE
CULTURE” is my own work conducted under the supervision of Mr. Anup
Karmakar, Assistant Professor, Devleela Lifesciences Pvt. Ltd. , has been
approved by the Shri Rawatpura Sarkar University, Dhaneli Raipur
Chhattisgarh. I have put 30 days attendance with the supervisor at the
Research lab.

I further declare that to the best of my knowledge this thesis does not contain
any part of any work which has been submitted for the award of any degree
either this university or any other university/Deemed university without proper
citation.
Mr. Anup Karmakar
Rajdeep Bose
Assistant Professor Signature of thecandidate
Signature of the supervisor
Mr. Rajendra Surana
Director
(Devleela Lifesciences Pvt. Ltd.)

2
 DECLARATION ON PLAGARISM
I declare that all materials in my thesis entitled “ANTIBIOTIC SCREENING FOR
ELIMINATION OF CONTAMINATION BACTERIA IN BAMBUSA BALCOOA TISSUE
CULTURE” is my own work and do not involved plagiarism. I also certify that:
a) No sentence, equation, diagram, table, paragraph, or section has been
copied verbatim from previous work unless it is placed under quotation marks
duly referenced.
b) The work presented is original and it is my own work. No ideas, processes,
results, or words of others have been presented as my own work.
c) Report on plagiarism check is appended.

Place: Raipur
Rajdeep Bose
Date: Signature of the candidate

3
 ACKNOWLEDGMENT

I extend my gratitude to the individual and organization who collaborated with

me on this project. I would like to thanks Devleela lifesciences, pvt. Ltd., for
their willingness to collaborate and their valuable inputs throughout the
project. Their expertise and contribute have significantly enhanced the quality
of this work.
I would like to thanks Mr. Rajendra Surana, The Director, Devleela Lifesciences
Pvt. Ltd., Raipur (CG), for giving me permission to commence my work in their
facility.
I would like to acknowledge the support received from Mr. Anup Karmakar,
scientist, Devleela pvt. Ltd., my supervisor for providing the necessary
resources and encouragement that enabled this collaborative effort.
I am thankful to all the lab members, especially Ms. Yamuna, lab assistant
and all workers of Devleela Lifesciences for providing me all the support and
facilities in the laboratory.
I feel short of word to express my deep sense of gratitude and indebtedness to
Dr. Surendra Gautam, HoD Lifeciences, and Dr. Anubhuti Koshle, Dean,
faculty of science at Shri Rawatpura Sarkar University, Dhaneli, Raipur (C.G)
for their scholarly guidance, endless patience and impressive supervision to
complete the project work successfully.
I am also grateful to my fellow students who worked alongside me, sharing
their knowledge and skill. Our collaboration fostered a creative and
productive environment, leading to the successful completion of this project.
The heavenly grace of almighty has defined and given meaning to my
existence. My heart brims with gratitude for my parents and whole family for
their perpetual blessing, inspiration, love and support.
-Rajdeep Bose

4
 Abbreviations

IAA – Indole acetic acid.

A naturally occurring auxin that promotes cell division, elongation, and root
initiation. It is often used in lower concentrations compared to other auxins
due to its higher potency.

BAP -Benzyl amino purine

A cytokinin that promotes cell division, shoot proliferation, and bud break. It is
often used in combination with auxins to regulate the balance between shoot
and root development.

PVP -Polyvinyl pyrrolidone.

A synthetic auxin with similar effects to IBA, but often used at even lower
concentrations due to its high activity. It can be particularly effective for
promoting rooting in difficult-to-root plants.

IBA -Indole butyric acid

Another auxin commonly used for root initiation and development. It is
generally considered more effective for rooting than IAA, especially for woody
plants.

EDTA -Ethylene diamine tetra acetic acid.

5
 CONTENT TABLE

Content Page no.

Introduction

Review of literature

Materials and methods

Result

Discussion

Summary

Bibliography

6
 INTRODUCTION
Plant tissue culturing techniques have become especially important in the
agricultural community over the past 10 years. During this time period, plant
tissue culture has effectively moved from the confines of small laboratories
and has taken its place among some of the more mainstream, broad-scale
techniques employed by the agriculture industry.
Plant tissue culture, more technically known as micropropagation, can be
broadly defined as a collection of methods used to grow large numbers of plant
cells, in vitro, in an aseptic and closely controlled environment. This technique
is effective because almost all plant cells are totipotent—each cell possesses
the genetic information and cellular machinery necessary to generate an entire
organism. Micropropagation, therefore, can be used to produce a large number
of plants that are genetically identical to a parent plant, as well as to one
another.
Bambusa balcooa is a symbodial bamboo species, grows well in humid tropical
and subtropical regions of the Indian sub continent with a long period of
vegetative growth followed by seedling and death of the clamp. It produces
clumps upto 10 to 15 m height and 4 to 8 cm dia. In India it is distributed
naturally in the North and North- Eastern parts of the country. It is a tall, sturdy
and quick growing bamboo suitable for the production of high quality paper and
furniture.
1. It is reported that the succulent shoots of Bambusa tulda are rich in
phytosterols and the fermented shoots used for the production of sterol
drugs.
2. It occurs in moist alluvial flat land along water courses upto an altitude
of 1500 m. in finer textured soils in the semi evergreen forests in relatively
low rainfall areas in Assam. This species contained high reserve of
organic matter, nitrogen, calcium, potassium and phosphorus under the
soils where it grows.

7
3. Records of bamboo flowering is limited in the literature due to
monocarpic nature and the unusually long flowering cycle.
4. It is also found that flowering cycle is reported to vary from 30 to 60 years.
The maturation of the tree species affects the potentiality of auxiliary
buds and it is reported that the success of clonal multiplication from
adult culm is restricted by many factors.
5. However, the reports on clonal multiplication from adult bamboos are
very limited.
6. The sexual method of reproduction is not reliable because of long and
erratic flowering cycle, short viability of seeds and consumption of seeds
by wild animals. Also propagation by vegetative means is difficult on
account of fewer and bulky propagules and season specificity. So, tissue
culture is the only convenient method for large scale production.
Tissue Culture research on bamboo was reported by Alexander and
Rao16On embryo Culture of Dendrocalamus strictus and since Then
starting from Huang and Murashige17 A Good start has been made on tissue
culture and A number of laboratories have begun to make Plant species. In
the earlier records on tissue culture, the Initiation of the culture was done
through seed And maximum rooting developed (92 per cent) Occurred in 3
weeks. The survival percentage Was recorded 80 to 90 per cent.18,19In the
Present research, an attempt was made to Report for mass multiplication of
Bambusa balcooa, an economically important bamboo Species. The
protocol developed here is Successful for commercial production the
Species through auxiliary bud culture. Survival Rate was recorded 100 per
cent after plantation In the field. This type of commercial Production of
bamboo is the first attempt in the North Eastern part of India through tissue
Culture process. The survival rate is 100 per Cent also first record in this type
of study.

8
 REVIEW OF LITERATURE
PLANT TISSUE CULTIRE:-
Plant tissue culture is a laboratory technique that involves the aseptic
cultivation of plant cells, tissues, or organs in a controlled environment. This
method allows researchers and horticulturists to propagate plants with
specific characteristics and study various aspects of plant growth and
development.

Introduction of Bambusa balcooa

Bambusa balcooa is a symbodial bamboo species, grows well in humid tropical
and subtropical regions of the Indian sub continent with a long period of
vegetative growth followed by seedling and death of the clamp. It produces
clumps upto 10 to 15 m height and 4 to 8 cm dia. In India it is distributed
naturally in the North and North- Eastern parts of the country. It is a tall, sturdy
and quick growing bamboo suitable for the production of high quality paper and
furniture. India is the second-largest bamboo-growing country in the world,
after China. It has 13.96 million hectares of bamboo, which is about 13% of the
country’s total forest area. India’s annual bamboo production is around 4.6
million tonnes, with about 1.9 million tonnes used by pulp industries.

Process Involves in Tissue Culture of Bambusa balcooa:-

Commonly we use micro propagation for the process of tissue culture of
Bambusa balcooa. Here is an insight introduction for the process of
micropropagation.

Micropropagation:-
Micro propagation, also known as tissue culture propagation or in vitro
propagation, is a plant propagation technique that involves the aseptic culture

9
of plant cells, tissues, or organs in a nutrient medium to produce multiple
identical copies of the parent plant, known as clones.
Micro propagation of plants need several elements to complete the process,
the key elements are as follow –
· Explants
· Culture media
· Aseptic conditions
•Culture vessels
· Hormones/plant growth regulators (PGRs)

· Equipment
· pH and temperature control
· Subculturing techniques
· Rooting and acclimatization

Explants

An explant in the context of plant tissue culture refers to a small piece of plant
material taken from a parent plant and used as the starting material for
initiating a culture. The choice of explant is crucial as it determines the type of
tissues or organs that will be cultured and the subsequent growth and
development of the cultured cells. Explants can be obtained from various parts
of the plant, and the selection depends on the specific goals of the tissue
culture process.
Some types of explants are –

10
 • Shoot Tips: The apical meristem or shoot tips contain actively dividing
cells and are commonly used for micropropagation. They can give rise to
multiple shoots when cultured in a suitable medium with cytokinins.
• Callus: Callus tissue, which is a mass of undifferentiated cells, can be
used as an explant. Callus can be induced from various plant tissues and
is often used as an intermediary in regeneration processes.

The choice of explant depends on factors such as the plant species, the
purpose of the tissue culture, and the desired outcome.

Culture Media:
The culture medium is a nutrient-rich agar or gel-based substance containing
essential nutrients, vitamins, sugars, and growth regulators.
The endophytic contaminant (present within the explants) is not easily
controlled. Endophytic fungus could be controlled by using systemic
fungicides but controlling bacteria is again more troublesome. Antibiotic with
broad spectrum activity coupled with low phytotoxicity is prerequisite to get
better results. Treatment duration and type of antibiotic are the critical factor
to reduce the contamination. But unscientific use of antibiotic may lead to the
development of resistant microbial strains. That is why antibiotic selection
after identification of the contaminants may be an efficient way to counter this
problem.

BIOTIC CONTAMINATION IN PLANT TISSUE CULTURE

Microbial contaminants may arises from different sources like infected plant
materials, improper tissue culture technique and poor laboratory condition.
Explant contamination is related to several factors like source of explants and
environment. Fungal and bacterial contamination is one of the serious

11
problems for the micro-propagation of woody plant species. Among fungal
contamination, presence of systematic fungal contamination is the most
problematic issue of micropropagation of mature woody species. The fungal
contamination was considered as most predominant factor
in B.Balcooa during tissue culture. In plant like bamboo, the nodal explants
have large intercellular spaces, which are exposed during cutting before
surface sterilization and promote the entry of bacterial and fungal spores into
explant. As a result they are unable to control by the surface sterilization
protocol and further found as contamination advance stages in medium.
Presence of this microorganisms leads to increase of plant mortaltity, create
variation in growth (reduction of shooting proliferation and rooting), tissue
necrosis even plant death.
Bacterial contamination is easily detected by visual inspection of the culture
within a few days of it becoming infected;

• Infected cultures usually appear cloudy (i.e., turbid), sometimes with a

thin film on the surface.
• Sudden drops in the pH of the culture medium is also frequently
encountered.
• Under a low-power microscope, the bacteria appear as tiny, moving
granules between the cells, and observation under a high-power
microscope can resolve the shapes of individual bacteria.

EPIPHYTIC VS ENDOPHYTIC
The contaminant in culture media may have immediate or latent expression in
which it remains dormant for long time. Epiphytic bacteria live on plant
surfaces and can be removed through chemical disinfectants. In contrast, the
endophytic microbes i.e., microbes that colonize living, internal tissues of
plants, without causing any immediate negative effect and are not easily

12
eliminated by simple surface-sterilization methods. For this reason the existing
contaminants are generally controlled by using either antibiotic or fungicide.
That is why the antibiotic therapy is getting much importance to solve this
problem. Now a day, there are several alternative ways to control the
contaminants reported by several authors such as through light and heat,
micro wave and hot water treatment. But till now most researchers depends on
chemicals to control the contaminants during in vitro propagation of plant.

SELECTION OF ANTIBIOTIC
Elimination of the latent contamination in culture may be done by using several
antibiotics. But type, application and treatment duration of antibiotics vary
plant to plant . In general antibiotics are broadly classified into two groups i.e;
bactericidal (kills bacteria) and bacteriostatic (prevent bacterial growth).
Several researchers experimentally proved that the uses of bactericidal are
more advantageous over bacteriostatic antibiotic to control the endophytic
contamination if there is no side effect of antibiotics in explants. An ideal
antibiotic in such case should be attributed as stable, easily soluble in nature,
not affected by components and pH of the medium, lacks side effects, broadly
active, non-resistance inducing, inexpensive and non-toxic to humans. But
tremendous use of antibiotics is not recommended because of its phytotoxicity
and selection for resistance strains. Since most of antibiotics have narrow
target range for bacteria, combination of antibiotics were found better to
reduce contamination as well as damaging plants due to their synergistic
effect. But antibiotic having broad range of target may shown better response
than the combination of antibiotics.

Similar to antibiotic, idle fungicide should be nontoxic to plant cell and broad
spectrum fungicidal activity. Since continuous use of single antibiotic often
leads to antibiotic resistant microbial contaminants on culture, combination of
different antibiotics is better option to counter the problem. This approach if
suitable to solve the problem then there is a need of modification in

13
concentration of antibiotics to lowering down the phytotoxicity level of
antibiotic on plant since effective concentration for both antibiotic may affect
the plant. Combination of antibiotics will kill contaminants without damaging
the plant. Use of combination of bactericides in bamboo/woody plants tissue
culture, which has limited uses of antibiotics, may be propoted to avoid the
unwanted effect of microbial contaminants on the growth of plant.

Aseptic Conditions:
Sterile conditions are maintained throughout the entire tissue culture process
to prevent contamination by microorganisms. Techniques include working in a
laminar flow hood and sterilizing equipment, media, and containers.

Culture Vessels:
These are containers in which plant tissues are cultured. They can be glass or
plastic and come in various shapes and sizes. The vessels must be autoclaved
or otherwise sterilized before use.

Equipment:
Essential equipment includes laminar flow hoods, autoclaves for sterilization,
incubators for maintaining optimal temperature and lighting conditions, and
microscopes for monitoring cell and tissue development.

pH and Temperature Control:

pH levels and temperature are critical factors influencing the success of plant
tissue culture. pH is usually maintained around 5.7, and temperature is
controlled based on the specific requirements of the plant species.

14
Subculturing Techniques:
Periodic transfer of the cultured tissues to fresh media to prevent nutrient
depletion and maintain the health of the cultures.

Rooting and Acclimatization:

Once shoots have developed, they may need to be transferred to a rooting
medium to stimulate root formation. After rooting, plants are gradually
acclimatized to normal environmental conditions before being transferred to
the field. The acclimatization involves two steps as primary hardening and
secondary hardening.

The role of primary hardening –

1. The plantlets are initially placed in controlled environment with

regulated temperature, humidity and reduced light intensity. This help
in reducing plant stress from transition.
2. With time the plants are slowly exposed to external condition by
increasing light intensity and fluctuating temperature and humidity
level.
3. With maintaining cleanliness and aseptic techniques it also involves
the reduction of sterility maintained during tissue culture which help
plant in adapting less controlled environment.

The role of secondary hardening –

15
 1. In shade house plants experience more direct and increased exposure
to natural sunlight. This allows them to adapt to higher light intensities,
preparing them for transplantation into open fields or gardens.
2. Unlike the controlled conditions of primary hardening, the transitional
environment during secondary hardening provide less environmental
control. This includes exposure to fluctuating temperatures, varying
humidity levels, and a more natural day-night cycle.
3. Secondary hardening aims to enhance the stress resistance of tissue-
cultured plants by providing them to conditions closer to those found
in their final growing environment. This contributes to the development
of hardier and more resilient plants.

Recent challenges In plant tissue culture:

High initial investment: Setting up a tissue culture lab requires significant

investment in equipment and expertise.
Soma clonal variation: Slight genetic variations can occur during the culture
process, requiring care and selection of true-to-type plantlets.

Hardening and field establishment: Successful acclimatization and

adaptation to field conditions are crucial for plant survival and performance.
Technical expertise: plant tissue culture is a complex process that requires
skilled personnel to handle the sterile environment and manipulate plant
tissues.

STREAKING PLATE METHOD:-

Streaking is a technique used in microbiology for the isolation of single
colonies of microorganisms, either from a mixed species or from the same
species. This technique is mostly applicable to bacteria but is also used for

16
some yeasts. It is an old technique that has been in use since the time of Rober
Koch. It was first demonstrated by Loeffler and Gaffky in Koch’s laboratory.

Streaking Plate Method Principle:-

The streak plate method is based on the principle of dilution. It can be
described as a rapid qualitative isolation technique. The main criterion of
isolation is to obtain a reduced number of colonies. In this technique, a loopful
of culture is spread on an agar plate to get individual cells far apart enough from
each other. The streaking method gradually dilutes the inoculum such that the
bacterial cells can be counted as colony forming units (CFUs).

Types of Streak Plate Method:-

1. Quadrant Streaking: This is the most common method of streaking, where
the petri dish is divided into four quadrants and then inoculated. It is also
known as a four-quadrant streak. A loopful of inoculum is taken and streaked
such that the first quadrant contains the highest concentration of the
inoculum, followed by the second quadrant, third quadrant, and fourth

quadrant.

17
This is a discontinuous method where the loop is sterilised after streaking in
every quadrant. By the time the fourth quadrant is streaked, the inoculum is
diluted enough to give rise to individual colonies.

2. T-Streaking: In this method, the petri dish is divided into three quadrants by
drawing the letter ‘T’. Similar to four-quadrant streaking, it is a discontinuous
method where each quadrant is streaked after loop sterilisation, and the

quadrant that is streaked last gives isolated colonies.

3. Continuous Streaking: In this type of streaking, the inoculum is spread
from one edge to the centre of the plate. The plate is rotated 180°, and the

remaining portion is streaked without sterilising the loop.

Another form of continuous streaking is used for diagnostic purposes. The

petri dish is divided into sections, and different cultures, such as urine,
sputum, pus, etc., are streaked in each section to get maximum output.
4. Radiant Streaking: In this streaking method, the inoculum is spread on one
edge of the petri dish. From the edge, vertical lines are streaked in the upward

18
direction. Next, the vertical lines are streaked horizontally to obtain pure
isolated cultures.

Streak Plate Method Procedure:-

• Sterilise all the instruments, flasks and media that are required for the
streaking procedure.
• Clean your work area using a disinfectant to minimise any
contamination.
• Set up the bunsen burner in your work area carefully.
• Wash your hands with an antiseptic solution before handling any
microbial solution.
• Label the petri dish with all important information, such as your name,
date, media used and the culture being inoculated.
• To pick up the sample, you can use either a metal loop or disposable
plastic loops.
• A loopful of sample is streaked on the first quadrant in a back-and-forth
motion on the agar plate.
• Sterilise the loop by heating it in the bunsen burner if using a metal loop.
• Streak the other three quadrants by a similar method.
• Close the lid of the plate after streaking, and store the dish upside down
in an incubator with optimal temperature.

Advantages of the Streak Plate Method

• It is one of the most popular techniques used to obtain isolated
colonies of bacteria.
• It finds a great application in biotechnology as it can be used to identify
transformed bacteria from non-transformed bacteria by adding an
antibiotic to the growth medium.
• It also finds great application in diagnostic purposes.

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GRAM STAINING
Gram stain is a laboratory test that checks for bacteria at the site of a
suspected infection or in certain bodily fluids. A medical laboratory scientist
processes the Gram stain, which gives relatively quick results, so healthcare
providers can know if bacteria are present, and, if so, the general type(s). This
can help guide further identification tests and treatment options.

Bacteria are a large group of one-celled organisms. They can live in different
places in your body and on your skin. While some types of bacteria are
harmless or even beneficial, others can cause infections and disease. A Gram
stain helps diagnose harmful bacteria.

Under a Gram stain, different kinds of bacteria change one of two sets of
colors (pink to red or purple to blue) under a special series of stains and are
categorized as “gram-negative” or “gram-positive,” accordingly. Gram staining
works by differentiating bacteria by the chemical and physical properties of
their cell walls.

However, not all forms of bacteria can be tested using the Gram stain method,
and Gram stains don’t usually provide a diagnosis alone. Instead, they help to
broadly determine the type of bacteria.

Gram staining is an essential staining technique in microbiology that

scientists have used for hundreds of years. It’s named after Danish
bacteriologist Hans Christian Gram, who first introduced it in 1882, mainly to
identify organisms causing pneumonia.

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GRAM-POSITIVE ORGANISMS:-
Gram-positive bacteria have cell walls that contain thick layers of
peptidoglycan, a substance that forms the cell walls of many bacteria. The
peptidoglycan forms about 90% of the cell wall in gram-positive bacteria. This
causes them to appear blue to purple under a Gram stain.

Gram-positive organisms include:

• Staphylococcus species.
• Streptococcus species.
• Corynebacterium species.
• Clostridium species.
• Listeria species.

GRAM-NEGATIVE ORGANISMS:-
Gram-negative bacteria have cell walls with thin layers of peptidoglycan (10%
of the cell wall) and high lipid (fatty acid) content. This causes them to appear
red to pink under a Gram stain.

Gram-negative organisms include:

21
 • Neisseria gonorrheae and Neisseria meningitides.
• Moraxella species.
• Escherichia coli (E. Coli).
• Pseudomonas species.
• Proteus species.
• Klebsiella species.

GRAM STAINING PROCEDURE:-

At the lab, a medical laboratory scientist smears or spreads the sample
on glass microscope slides. These slides are known as smears. They then
apply a series of stains to the smear to perform a Gram stain.
The Gram staining process includes four basic steps, including:

• Applying a primary stain (crystal violet).

• Adding a mordant (Gram’s iodine).
• Rapid decolorization with ethanol, acetone or a mixture of both.
• Counterstaining with safranin.

MATERIALS AND METHODS

Requirements for Plant Tissue Culture

22
Culture Room:
In plant tissue culture, a culture room is a dedicated space designed to
maintain aseptic conditions for the growth and propagation of plant cells,
tissues, or organs. The culture room typically incorporates laminar flow
hoods, sterile workstations, and controlled environmental conditions,
including temperature, humidity, and light cycles. These conditions are
optimized based on the specific requirements of the plant species being
cultured to ensure successful tissue culture experiments and the production
of healthy plant material.

Growth Room:
The growth room in plant tissue culture is a controlled environment where
plant tissue cultures are cultivated and nurtured to promote their growth and
development. Temperature, light intensity, and photoperiod are carefully

23
regulated to simulate the natural conditions necessary for plant growth.
Growth rooms are crucial for the multiplication of plant cultures, callus
induction, and the development of shoots and roots in vitro.

Washing Area:
The washing area in plant tissue culture laboratories is dedicated to the
cleaning and sterilization of glassware, instruments, and other materials used
in tissue culture procedures.
Maintaining cleanliness and sterility is essential to prevent contamination,
which could adversely affect the integrity of the
plant tissue cultures. The washing area typically
includes sinks, autoclaves, and designated
spaces for decontamination procedures.

Greenhouse:
In plant tissue culture, a greenhouse provides a
controlled outdoor environment for the
acclimatization and hardening of plantlets
produced in vitro before their transfer to the
natural environment. The greenhouse regulates temperature, humidity, and
light conditions to gradually expose theplants to external factors. This helps in
the successful transition of tissue-cultured plants from sterile laboratory
conditions to the field.

Shade House:

24
A shade house is a facility used in plant
tissue culture to provide a controlled
environment with reduced light intensity
for shade-loving or light-sensitive plant
species. It is particularly useful for
acclimatizing plants that have been
propagated in vitro to lower light levels
before transplanting them into outdoor
conditions. The shade house protects
plants from excessive sunlight, allowing
for a gradual adaptation to ambient light
conditions.

Instruments and Equipment required

Laminar Air Flow Cabinet (LAFC):

Principle: Laminar air flow works on the principle of directing air in a single,
continuous stream, usually in a vertical or horizontal flow, to maintain a sterile
environment. This minimizes the risk of airborne contamination during
sensitive procedures.

25
 Working: A laminar flow
hood consists of a high-
efficiency particulate air
(HEPA) filter that removes
particles from the incoming
air. The filtered air then
moves in a laminar, uniform
flow over the work surface,
creating a clean area for
tasks such as cell culture
or sample preparation.

Autoclave:
Principle: Autoclaves use steam under high pressure to achieve sterilization.
The high temperature and
pressure result in the
destruction of bacteria,
spores, and other
microorganisms, ensuring
the complete elimination of
potential contaminants.
Working: Items to be
sterilized are placed inside
the autoclave, and steam is
generated by heating water.
As the pressure increases,
the temperature of the
steam rises, effectively
sterilizing the contents. The
autoclave then allows for a

26
controlled cooling process before items are safely removed.

Incubator:

Optimal plant growth requires precise control of temperature and humidity.

Incubators provide a controlled environment that mimics ideal conditions for
plant development by maintaining constant and specific temperatures and
appropriate humidity levels within the chamber.

Culture Bottles:

Principle: Culture bottles provide a sterile and controlled

environment for the growth of microorganisms or cells. The choice
of material (glass or plastic) depends on the type of culture and
the specific needs of the experiment.
Working: Culture bottles are filled with a nutrient-rich medium
supporting the growth of cells or microorganisms. The sealed
environment prevents contamination while allowing observation
and manipulation of the culture through the bottle’s closure.

Forceps and Scalpel:

Principle: Forceps are designed for delicate grasping and
manipulation of small objects, while scalpels are sharp cutting
instruments used for precise dissections and sample preparation.

Working: Forceps are operated by hand, allowing researchers to

handle small items with precision. Scalpels are used to make
incisions or separate tissues, aiding in various laboratory
procedures, including dissections and the collection of biological
samples.

27
pH meter:
Principle: pH meters measure the concentration of hydrogen ions in a
solution, indicating its acidity or alkalinity. The pH scale ranges from 0 to 14,
with 7 being neutral, below 7 acidic, and above 7 alkaline.
Working: A pH meter
consists of an
electrode that
measures the electrical
potential difference
between itself and a
reference electrode in
the solution. This
potential difference is
then converted into pH
readings, providing
accurate information
about the solution’s
acidity or alkalinity.

Each tool plays a vital role in maintaining sterility and providing optimal
conditions for plant regeneration and development.

28
 METHODOLOGY

Here are some steps for antibiotic screening to eliminate bacterial

contamination in Bambusa balcooa plant tissue culture (PTC):

1. Add antibiotics to the multiplication medium.

2. Test the antibiotics’ effectiveness.
3. Identify an antibiotic and its MIC value.
4. Apply the antibiotic to the bacterially contaminated tissue.
5. Allow the tissue to outgrow the contamination.

Antibiotics that can be used to inhibit bacterial contaminants include:

Tetracycline, Streptomycin, Vancomycin, Rifampicin, Gentamycin,
Cefotaxime.
These antibiotics can be used in combinations to achieve better results.

The agar embedding method is another method for eliminating bacterial

contamination. This method involves placing contaminated moss tissue in
contact with an antibiotic that inhibits bacterial growth.
The disk diffusion method and E test can also be used to identify an antibiotic
and its MIC value. These methods can eliminate bacterial contaminants while
causing minimal damage to explants.

29
To avoid contamination from explants before being used in PTC, different
sterilizing agents can be used. Some examples include:

• 50% Chlorox plus 0.1% mecuric chloride

• 50% chlorox
• 0.1% Mecuric chloride
• 70% Ethanol

1. Add Antibiotics to the multiplication medium.

Antibiotics can be added to the multiplication medium after

autoclaving. If the antibiotic’s melting point is higher than the
sterilization temperature, it can be added before sterilization. After
sterilization, the antibiotic must be filtered through a 0.2 um filter.

2. Test the Antibiotics effectiveness.

To test the effectiveness of antibiotics in eliminating bacterial

contamination in plant tissue culture (PTC), antibiotics are added to the
multiplication medium. The antibiotics are added in different dosages,
both alone and in combinations.

The inhibition zone surrounding each antibiotic disk is measured to the

nearest millimeter.

Agar disk-diffusion testing is the official method used in many clinical

microbiology laboratories for routine antimicrobial susceptibility
testing.

Antibiotics are often incorporated as prophylactics in the tissue culture

medium or are used to suppress or eliminate bacteria once a
contaminant is detected.

30
 3. Identify an antibiotic and its MIC value.

MIC is the lowest concentration of an antibacterial agent expressed in

mg/L (μg/mL) which, under strictly controlled in vitro conditions,
completely prevents visible growth of the test strain of an organism.

MIC Determination Methods:-

• Dilution method.

1.In agar

2.In a liquid medium

• Gradient method.

Strips impregnated with a predefined concentration gradient of

antibiotic.
4. Apply the antibiotic to the bacterially contaminated tissue.

Antibiotics can be used to suppress or eliminate bacteria in plant tissue

cultures. They can be incorporated into the tissue culture medium as
prophylactics, or used once a contaminant is detected.

Antibiotics can be applied to bacterially contaminated tissue using the

agar embedding method. This method involves placing the
contaminated tissue in contact with an antibiotic that inhibits bacterial
growth.
Antibiotics can also be placed on the surface of Mueller-Hinton agar
(MHA) medium that has been inoculated with bacterial contaminants.

Antibiotics can be effective in controlling bacterial contamination in explants

and culture media. For example, cefotaxime can be used alone or in

31
combination with other antibiotics. Streptomycin plus gentamicin, as well as
cefotaxime and gentamicin, have also yielded some clean explants.

However, antibiotics will not penetrate tissues in sufficient concentrations to

kill endogenous pathogens. Antibiotics can help prevent bacteria from
overgrowing the plant nutrient medium
5. Allow the tissue to outgrow the contamination.

Production Process of Tissue Culture of Bambusa balcooa

32
The tissue culture protocol for bamboo is very well standardized in India. The basic steps
involved in the production of tissue culture bamboo are as follows:

a. Selection of a superior mother clone

b. Isolation of ex-plant
c. Inoculation and Incubation of ex-plant
d. Initiation of callus
e. Subculturing
f. Regeneration (multiplication and rooting)
g. Primary hardening (Green house)
h. Secondary hardening (Shade house)
i. Field transfer

33
Inoculation and Incubation:-

After the primary sterilization the ex-plant is transferred to inoculation room

and now dipped in 0.5% mercuric chloride for 1 hour, after that it is washed
with water.

The sterilized explants are then inoculated onto a carefully prepared culture
medium containing essential nutrients and plant growth regulators (PGRs).
The specific composition of the medium and PGRs are tailored to induce
callus formation and subsequent plant development. The inoculated cultures
are then incubated under controlled conditions of temperature, light, and
humidity to promote optimal growth and development.

The media we use will be Murashige and Skoog (MS) medium.

The MS media is prepared in lab with the help of different chemical

components as:

34
 Component table of MS Media

Constituent Quantity/litre
Ammonium Nitrate 1.65g
Potassium Nitrate 1.9g
Calcium Chloride 0.44g
Meso-Inositol 0.1g
Magnesium 0.370g
Sulphate
Manganese 0.223g
Sulphate
Zinc Sulphate 8.6g
Cupric Sulphate 0.025g
Ferrous Sulphate 27.8g
Sodium EDTA 37.8g
Potassium 0.17g
Dihydrogen
Potassium Iodide 0.83g
Boric Acid 6.2g
Sodium Molybdate 0.25g
Cobalt (II) chloride 0.025g
Adenine Sulphate 0.18g
Thiamine HCL 0.1g
Nicotinic acid 0.5g
Pyridoxine HCL 0.5g
Glycine 2g
Sucrose 30g
Agar-Agar 7g

For Multiplication Media

BAP 4g
IAA 1g

35
 For Rooting Media

PVP 0.5g
IBA 1g
Charcoal 0.25g

36
Charcoal - Importance of charcoal are:

Adsorption: It adsorbs phenolic compounds and other inhibitory substances that can be
produced by explants, improving their growth and development.

pH buffering: It helps to maintain a stable pH in the culture medium, which is crucial for
optimal plant growth.

Microbial control: It can suppress the growth of bacteria and fungi in the medium, reducing
the risk of contamination.

The steps of media preparation involves –

a. Weighing of all the chemicals

b. Mixing with the water
c. Correcting the pH of the solution to 6.8 using NaOH and HCl
d. Heating the solution and mixing well
e. Addition of agar at the boiling mixture
f. Pouring the media in culture bottle and refrigerate.

Based on need of media, media are of two types as- multiplication media and rooting
media.

A. Multiplication media- consists of Indole-3-acetic acid (IAA) and N6-Benzyl

amino purine. It support cell multiplication.(white coloured)
B. Rooting media- consists of Polyvinyl pyrrolidone (PVP), Indole-3-butyric acid
(IBA) and Charcoal. It support the rooting of plant.(black coloured)

After the cooling the media is ready for the culture addition.

The cutting of the white part of the sterilized ex-plant is now dipped in media.

37
5.Subculturing

When optimum growth occur sub culturing is done. In this process plant tissue are
removed from media after two week interval and are cut into small pieces. Then this small
pieces are transferred to another bottle containing same medium as in initiation of culture.
The bottle cap are tapped then the explant transferred to shoot multiplication.

6.REGENERATION

Following controlled manipulation of PGRs in the medium, shoots and roots begin to
develop from the callus mass. This marks the crucial regeneration stage, where the
undifferentiated callus undergoes organogenesis, forming the miniature plantlets. After
each subculture the plantlets are sent to the growth room which provides them light for
photosynthesis with the intensity of 3000-4000 lux. The temperature of growth room is
maintained to be 24-26°C.

7.Primary Hardening (Greenhouse)

The newly formed plantlets, still fragile and adapted to the sterile laboratory environment,
require gradual acclimatization to harsher greenhouse conditions. This primary hardening
stage involves slowly reducing humidity levels and increasing light intensity, enabling the
plantlets to adjust to their new environment. This step takes about 30-35 days.

8.Secondary Hardening (Shade House)

Once sufficiently acclimatized in the greenhouse, the plantlets are further transferred to a
shade house, where they are exposed to conditions closer to their natural outdoor
environment. This secondary hardening phase toughens the plantlets, preparing them for
final transplantation into the field. The plants take 60 days in shade house for adaptation.

38
9.Field Transfer

Following successful hardening, the fully developed plantlets are finally transplanted into
the field, where they can flourish and reach their full potential. These plants will exhibit the
same desirable characteristics as the chosen mother plant, contributing to improved
agricultural productivity and the preservation of valuable genetic traits.

10.Transportation

The fully developed plants are now transported to their benefactor for various purposes.it
can be study purposes, research, commercial purposes, business purposes etc.

Conclusion
The common problem of bacterial growth around in vitro shoots in Bambusa balcooa
tissue culture is due to (1) Pantoea agglomerans and (2) Pantoea ananatis. This can be
resolved through treatment of B. balcooa shoots with kanamycin for 10 days. No
phytotoxicity appeared when shoots were treated with kanamycin (10 μg/ml) and the
multiplication rate of treated B. Balcooa shoots was found to be similar to that of healthy
plants. Streptomycin sulfate, at higher concentration, also inhibited bacterial growth during
micropropagation of B. balcooa.

In addition, shoots grown in streptomycin sulfate tended to be shorter and have stunted
leaves. Thus, the study provides us with a technique to identify and resolve bacterial
contamination of (1) Pantoea agglomerans and (2) Pantoea ananatis during in vitro culture
of B. balcooa.

39
 RESULT
In this project of topic ANTIBIOTIC SCREENING FOR ELIMINATION OF
CONTAMINATION BACTERIA IN BAMBUSA BALCOOA TISSUE CULTURE we
worked on different culture media compositions and observe their culture
establishment and contaminations.
The results of above objectives are:

S.No. Percent of Treatment Time Percent of

Culture Culture
contamination Establishment
1. 100 0.05%Mercuric 10 min 0
chloride
2. 90 0.05%Mercuric 15 min 10
chloride
3. 60 0.01%Mercuric 10 min 40
chloride
4. 20 0.01%Mercuric 15 min 80
chloride

Excision of Explants
Explants were successfully excised from mother suckers with the help of
sharp-edged blade under aseptic conditions.

Sterilization of Explants:
For surface sterilization were used for establishments of contamination free
cultures of Bambusa. Among the four treatments, 100% contamination was
observed when 0.5% mercuric chloride was used. Optimum sterilization time
was found to be 15% and showed 80% contamination free culture
establishment.

40
16

14

12

10

0
0.05% Mercuric chloride 0.05% Mercuric chloride 0.01% Mercuric chloride 0.01% Mercuric chloride

Time culture contamination culture establishment

Media Preparation and Sterilization:

Murashige and Skoog’s (MS) medium were prepared in aseptic condition using
stock solution and agar. Media was sterilized through autoclave properly. After
2 days storage, no contamination was found to be occurred.

Explants Initiation

41
The development of nodal ex-plant, Bambusa balcooa with different
concentration of plant growth regulators (BA 0.5-1.5).The treated nodal
cultures inoculated in the solid MS media. After 2-3 weeks highest shoot
development is found in MS media with BA concentration 1.5 mg/L i.e. 8.0±2.4
shoots per explant and highest shoot length 3.8±0.4 were recorded. Mature
buds were then transferred in multiplication media for 2-3 weeks at controlled
conditions.

Concentration Shoot
S.no. Media No. of shoot
mg/L length
1. MS+BA 0.5 3.8 ± 0.2 1.2 ± 0.1
2. MS+BA 1.0 5.0 ± 1.2 2.5 ± 1.3
3. MS+BA 1.5 8.0 ± 2.4 3.7 ± 0.4

42
9

0
MS + BA MS + BA MS + BA

conc. (mg/l) no. of shoots shoot length (cm)

Graph. Effect of BA on shoot multiplication

43
 DISCUSSION
Bambusa balcooa, commonly known as the female bamboo or
Indian bamboo, is a species of bamboo native to Southeast Asia and
the Indian subcontinent. It is a versatile and economically important
plant that has various uses and applications.

Bambusa balcooa invitro culture refers to the cultivation of this bamboo

species under controlled laboratory conditions, providing a controlled
environment for its growth and development. In vitro culture involves using
plant tissue culture techniques, where small plant parts, such as shoot tips or
nodal segments, are placed in a sterile nutrient medium to initiate growth. For
Bambusa balcooa, this technique holds immense potential in the propagation
and conservation of the species. It allows for the rapid multiplication of elite
and disease-free plants, ensuring genetic uniformity. Additionally, in vitro
culture facilitates the preservation of endangered or rare genotypes,
contributing to biodiversity conservation efforts. The controlled conditions
enable researchers to study and manipulate the growth and development of
Bambusa balcooa, offering insights into its physiology, biochemistry, and
genetics. This method can also be instrumental in the development of
improved varieties with desirable traits for various applications, such as
construction, agriculture, or environmental conservation. As technology and
research in plant tissue culture advance, the invitro culture of Bambusa
balcooa emerges as a promising avenue for sustainable bamboo cultivation
and resource management.

It is stated that tissue culture propagation ensures genetic stability. But it is

possible that continuous subculturing might result in soma clonal variation.
Research on this aspect will help to pinpoint the various aspects of this
phenomenon. If found advantageous, it might be possible to commercially

44
exploit this phenomenon. Certain novel approaches for improving banana
through tissue culture the achievement of which will definitely help in the
improvement of banana cultivation in India.

It is so concluded that bamboo can be grown in vitro as well as in vivo in case

to get some potential, bamboo are so far most consuming product through
the globe so its importance has given rise to the alternative methods of
propagation that is so called micro propagation. The give bolster to the
bamboo production to peak of its best quality round the world and will
available round the year to all country or even non producing countries as well
just by using the conventional method of propagation.Some uses of bambusa
are provided below:

Culinary Uses:

Some species of bamboo, including Bambusa balcooa, are edible. Discuss

the culinary uses of bamboo shoots and their nutritional value.

Construction Material:
Bambusa balcooa is known for its sturdy and straight culms. Discuss its use
as a construction material, such as in building houses, bridges, or fences.

Cultural Significance:
In many cultures, bamboo holds cultural and symbolic significance. Explore
any cultural or traditional uses of Bambusa balcooa in ceremonies, rituals, or
daily life.

Commercial Applications:

45
Bambusa balcooa has economic importance due to its use in various
industries. Explore its applications in furniture making, handicrafts, or paper
production.

Environmental Benefits:
Bamboo is known for its rapid growth and carbon sequestration capabilities.
Discuss how the cultivation of Bambusa balcooa can contribute to carbon
sequestration and sustainable forestry practices.

Research and Innovation:

Bamboo balcooa are used in research purposes where it is in vitro grown for
commercial as well as research purposes.

46
 SUMMARY

Over the past decade, plant tissue culture, specifically micropropagation, has
emerged as a crucial technique in bamboo cultivation, gaining prominence in
mainstream agricultural practices. This method involves growing plant cells in
a controlled, sterile environment, proving effective due to the totipotency of
almost all plant cells. India, a leading bamboo producer, has embraced tissue
culture for cultivating the balcooa variety, a preferred choice for its disease
resistance and high productivity.

Bambusa balcooa culturing involves the controlled cultivation of this bamboo

species in a laboratory setting through in vitro culture techniques. This
method utilizes plant tissue culture to propagate the bamboo from small plant
parts in a sterile nutrient medium. The culturing process allows for rapid
multiplication of elite and disease-free plants, ensuring genetic uniformity
and contributing to biodiversity conservation. It is particularly useful for
preserving rare or endangered genotypes. The controlled conditions of in vitro
culture enable researchers to study and manipulate the growth and
development of Bambusa balcooa, providing insights into its physiology,
biochemistry, and genetics. This method holds promise for the development
of improved bamboo varieties with desirable traits for applications such as
construction, agriculture, and environmental conservation. Overall, Bambusa
balcooa culturing presents a valuable approach for sustainable bamboo
cultivation and resource management.
In Conclusion, Presence of contaminants in culture affects the growth of
plant in vitro. Plant tissue culture is an expensive method, so contamination
frees cultures are need to be developed to get desirable profit. There several
antibiotics were already reported by several authors to eliminate the
endophytic contaminants during tissue culture of many plants due to failure
of general sterilization method. The endophytic contaminants are serious

47
concern in micropropagation. Presence of contaminants in culture affects the
growth of plant in vitro.
Plant tissue culture is an expensive method, so contamination frees cultures
are need to be developed to get desirable profit. There several antibiotics were
already reported by several authors to eliminate the endophytic contaminants
during tissue culture of many plants due to failure of general sterilization
method. Antibiotic as a media component is more effective as evident from
the report of earlier workers on different plants. But addition of antibiotic
within media may lead to phytotoxic effect on plant. So, broad spectrum
antibiotic with low phytotoxicity is only desirable to eliminate the endophytic
contaminants.
For this reason there need to find out the minimum inhibitory concentration of
antibiotic against particular microorganisms after its proper identification
using the most reliable molecular technique like 16S rDNA. There need further
research work particularly on bamboo since each antibiotic has different
mode of action, solubility etc.

48
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