I hereby declare that a dissertation work entitled ―Micropropagation studies on Bambusa tulda plant through nodal explant” Submitted to university in fulfillment for the award of degree in Bachelors Of Science (forestry) is carried out by me at State Research Institute Jabalpur Madhya Pradesh.

1. Plant Tissue Culture
Manzoor Nabi Wani
Only A Small Explant Is Enough To Get Millions Of Plants With Extremely High Multiplication Rate
2016
Every People In
India Depends
Directly On
Forests For
Sustenance.

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“Micropropagation Studies On Bambusa tulda”
Under The Supervision Of
(Sr. Scientist)
State Forest Research Institute Jabalpur Madhya Pradesh
Gangrar, Chittorgarh, 312901(Rajasthan)
Year 2016

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DECLARATION
I hereby declare that a dissertation work entitled ―Micropropagation studies on
Bambusa tulda plant through nodal explant” Submitted to university in fulfillment for
the award of degree in Bachelors Of Science (forestry) is carried out by me at State
Research Institute Jabalpur Madhya Pradesh.
Date:
Manzoor Nabi Wani
B.SC VII SEM

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ACKNOWLEDGEMENT
It is my pleasure to acknowledge the help which I received during the work and through
preparing thesis; this dissertation would not have been possible without guidance and
help of several individuals who contributed their valuable assistance in completion of
this study.
Foremost, I would like to express my sincere gratitude to the Director, SFRI
Dr.G.Krishna Murthy (IFS) for giving me an opportunity to be a part of institute.
I am greatly indebted to my Supervisor Dr.S.K.Tiwari, Scientist and Head, Forest
Genetics Plant Propagation and Biotechnology Division, SFRI, Jabalpur (M.P) for his
abiding interest and invaluable guidance.
I express my profound gratitude to Mr. Amit Panday, Senior Research Officer, Forest
Genetic Plant Propagation and Biotechnology Division, SFRI Jabalpur (M.P) whose
guidance helped me to proceed my work.
I am very thankful to my Dr. Vk.Solanki, Head of Department of Forestry and Mewar
University including S.K.Das and Dr.Ashok Gadiya for their precious guidance.
I shall be failing in my duty, if I do not gratefully acknowledge the valuable time and
inspiration given by, Mr. Maneesh Puri Goswami, Mr. Pankaj Saini and Mr. Vineet
Mhera, SFRI,Jabalpur, Whose tips provided me enthusiasm to work hard.
Finally, I must express my profound gratitude to my Family for providing unfailing
support. Last I would like to thank ALMIGHTY ALLAH for the blessings without which I
might not complete my work.
MANZOOR NABI WANI

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CONTENTS
S.No Topic Page No…
01 Introduction 05
02 Review of Literature 35
03 methodology 38
04 Result and Discussion 46
05 Conclusion 50
06 Reference 61
07 Photo gallery 65

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Introduction

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DESCRIPTION:
Biotechnology is a relatively new science with direct applications to the Agriculture
industry. This article describes some of the pros and cons of Biotechnology.
Introduction:
One of the newest, yet controversial fields in science today is biotechnology.
Biotechnology began in the 1970s after the development of genetic engineering that
allowed scientists to modify the genetic material of living cells. Genetic engineering is
the manipulation of DNA molecules to produce modified plants, animals, or other
organisms. DNA is the part of a cell that controls the genetic information of an animal or
plant. DNA is a double-stranded molecule that is present in every cell of an organism.
The genetic information is contained in individual units or sections of DNA called genes.
The genes that are passed from parent to offspring determine the traits that the
offspring will have. Scientists are now able to isolate the gene or genes for the traits
they want in one animal or plant and move them into another. The movement of a gene
from one organism to another is called recombinant DNA technology.
Gene: A unit of hereditary material located on a chromosome.
Genetic engineering: Movement of genes from one cell to another.
Hormones: Chemicals released by cells that affect cells in other parts of the body.
Only a small amount of hormone is required to alter cell metabolism.
Recombinant DNA technology:
An application of genetic engineering in which genetic information from one organism
is spliced into the chromosome of another organism.
Biotechnology:
Use of cells or components of cells to produce products or processes.
Definition of Plant Biotechnology:
In a broad sense:
Plant biotechnology covers many of the tools and techniques that are commonplace
in agriculture and food production.
In a narrow sense:
Biotechnology considers only the new DNA techniques, molecular biology and
reproductive technological applications, like gene manipulation, gene transfer, DNA
genotyping and cloning.

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DIFFERENT SCIENCE FIELDS CONTRIBUTING INTO THE
ADVANCEMENT OF BIOTECHNOLOGY.
Historical Advancement of Biotechnology:
Biotechnology related activities depend on two parameters: technological
advancement and knowledge of available biota. Technological upgradation goes
parallel with the over-all understanding of physical and chemical phenomenon in
different time periods. Hence, Biotechnology starts as early as human have realized the
importance of organism (animal/plants or microbes) to improve their life-style.
Important milestones of Biotechnology
S.No. Time
Period
Major break-through
1. 7000 BC-100CE
fertility.
fertilizer and insecticide respectively
2. Pre-20th Century

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for genetic traits.
3 20th Century
-D Struture of DNA.
-tube
baby.
4 21st Century
sequences.

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Applications of Biotechnology:
Biotechnology has influenced human life in many ways by inventions to make his life
more comfortable. Many scientific fields contribute to biotechnology and in return it
gives product for their advancement.
Impact of Biotechnology on different fields & human life.

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What is Plant Tissue Culture?
The aseptic culture of plant protoplasts, cells, tissues or organs under conditions which
lead to cell multiplication or regeneration of organs or whole plants ―
Definition: In plant tissue cultures, sterile plant material is cultured under aseptical
conditions in usually defined sterile culture medium often solidified by agar.
Commonly used terms in tissue culture:
Adventitious: development of organs such as buds, leaves, roots, shoots and
somatic embryos from shoot and root tissues and callus.
Agar: Natural gelling agent made from algae.
Aseptic technique: procedures used to prevent the introduction of microorganisms
such as fungi, bacteria, viruses and phytoplasmas into cell, tissue and organ cultures,
and cross contamination of cultures.
Autoclave: A machine capable of sterilizing by steam under pressure.
Axenic culture: a culture without foreign or undesired life forms but may include the
deliberate co-culture with different types of cells, tissues or organisms.
Callus: an unorganized mass of differentiated plant cells.
Cell culture: culture of cells or their maintenance in vitro including the culture of single
cells.
Chemically defined medium: a nutritive solution or substrate for culturing cells in which
each component is specified.
Clonal propagation: asexual multiplication of plants from a single individual or
explant.
Clones: a group of plants propagated from vegetative parts, which have been derived
by repeated propagation from a single individual. Clones are considered to be
genetically uniform.
Contamination: infected by unwanted microorganisms in controlled environment.

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Cryopreservation: ultra-low temperature storage of cells, tissues, embryos and
seeds.
Culture: A plant growing in vitro in a sterile environment.
Differentiated: cultured cells that maintain all or much of the specialized structure
and function typical of the cell type in vivo.
Embryo culture: In vitro culture of isolated mature or immature embryos.
Explant: an excised piece or part of a plant used to initiate a tissue culture.
Ex vitro: Organisms removed from tissue culture and transplanted; generally plants to
soil or potting mixture.
Hormone: Generally naturally occurring chemicals that strongly affect plant growth.
In Vitro: To be grown in glass.
In Vivo: To be grown naturally.
Laminar Flow Hood: An enclosed work area where the air is cleaned using HEPA
filters.
HEPA= HIGH EFFIECENCY PARTICULATE AIR
Medium: a solid or liquid nutritive solution used for culturing cells.
Meristem: a group of undifferentiated cells situated at the tips of shoots, buds and
roots, which divide actively and give rise to tissue and organs.
Micropropagation: multiplication of plants from vegetative parts by using tissue
culture nutrient medium.
Propagule: a portion of an organism (shoot, leaf, callus, etc.) used for propagation.
Somatic embryos: non-zygotic bipolar embryo-like structures obtained from somatic
cells.
Subculture: the aseptic division and transfer of a culture or portion of that culture to a
fresh synthetic media.

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Totipotency: Capacity of plant cells to regenerate whole plants when cultured on
appropriate media.
Transgenic: plants that have a piece of foreign DNA.
Undifferentiated: cells that have not transformed into specialized tissues.
Reasons for in vitro cultivation of plant material:
Controlled environmental conditions
Science, symbiotic orchid seed culture, metabolite production
Pathogen-free material
Once pathogen-free, the material propagated under sterile conditions remains
pathogen-free
Multiplication
Rapid clonal propagation; also done ex vitro for many plants
Embryo rescue (infertile hybrids)
Cryopreservation
Propagation and Distribution of Mutants, Colourmorphs, etc.
Possible also for ex vitro clonally propagated plants
Genetic engineering
True regeneration necessary, most meristems are not transformable.
History of plant tissue culture:
The science of plant tissue culture takes its roots from the discovery of cell
followed by propounding of cell theory. In 1838, Schleiden and Schwann
proposed that cell is the basic structural unit of all living organisms. They
visualized that cell is capable of autonomy and therefore it should be
possible for each cell if given an environment to regenerate into whole
plant. Based on this premise, in 1902, a German physiologist, Gottlieb
Haberlandt for the first time attempted to culture isolated single palisade
cells from leaves in knop‘s salt solution enriched with sucrose. The cells
remained alive for up to one month, increased in size, accumulated starch
but failed to divide. Though he was unsuccessful but laid down the
foundation of tissue culture technology for which he is regarded as the
father of plant tissue culture. After that some of the landmark discoveries
took place in tissue culture which are summarized as under:
- 1902 - Haberlandt proposed concept of in vitro cell culture
- 1904 - Hannig cultured embryos from several cruciferous species

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- 1922 - Kolte and Robbins successfully cultured root and stem tips
respectively
- 1926 - Went discovered first plant growth hormone –Indole acetic acid
- 1934 - White introduced vitamin B as growth supplement in tissue culture
media for tomato root tip
- 1939 - Gautheret, White and Nobecourt established endless proliferation of callus
cultures
- 1941 - Overbeek was first to add coconut milk for cell division in Datura
- 1946 - Ball raised whole plants of Lupinus by shoot tip culture
- 1954 - Muir was first to break callus tissues into single cells
- 1955 - Skoog and Miller discovered kinetin as cell division hormone
- 1957 - Skoog and Miller gave concept of hormonal control (auxin: cytokinin) of organ
formation
- 1959 - Reinert and Steward regenerated embryos from callus clumps and cell
suspension of carrot (Daucus carota)
- 1960 - Cocking was first to isolate protoplast by enzymatic degradation of cell wall
Plant Tissue Culture: Current Status and Opportunities 3
- 1960 - Bergmann filtered cell suspension and isolated single cells by plating
- 1960 - Kanta and Maheshwari developed test tube fertilization technique
- 1962 - Murashige and Skoog developed MS medium with higher salt concentration
- 1964 - Guha and Maheshwari produced first haploid plants from pollen grains of
Datura (Anther culture)
- 1966 - Steward demonstrated totipotency by regenerating carrot plants from single
cells of tomato
- 1970 - Power et al. successfully achieved protoplast fusion
- 1971 - Takebe et al.regenerated000000 first plants from protoplasts
- 1972 - Carlson produced first interspecific hybrid of Nicotiana tabacum by protoplast
- 1974 – Rein hard introduced biotransformation in plant tissue cultures
- 1977 - Chilton et al. successfully integrated Ti plasmid DNA from Agrobacterium
tumefaciens in plants
- 1978- Melchers et al. carried out somatic hybridization of tomato and potato resulting
in pomato
- 1981- Larkin and Scowcroft introduced the term somaclonal variation
- 1983 - Pelletier et al.conducted intergeneric cytoplasmic hybridization in Radish and
Grape
- 1984 - Horsh et al. developed transgenic tobacco by transformation with
Agrobacterium
- 1987 - Klien et al. developed biolistic gene transfer method for plant transformation
- 2005 - Rice genome sequenced under International Rice Genome Sequencing
Project.
Terminology in plant tissue culture:
Callogenesis: Callus Formation

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Embryogenesis: Somatic Embryo Formation
Organogenesis, Caulogenesis: Shoot Formation
Rhizogenesis: Root Formation
Types of Culture.
 Callus culture
 Suspension culture
 Embryo culture
 Endosperm culture
 Meristem culture
 Protoplast culture
Stage of plant tissue culture:
Stage 0: Preparation of donor plant. Any plant tissue can be introduced in vitro.
To enhance the probability of success, the mother plant should be ex vitro cultivated
under optimal conditions to minimize contamination in the in vitro culture.
Stage I: Initiation stage.
In this stage an explant is surface sterilized and transferred into nutrient medium.
Generally, the combined application of bactericide and fungicide products is suggested.
The selection of products depends on the type of explant to be introduced. The surface
sterilization of explant in chemical solutions is an important step to remove
contaminants with minimal damage to plant cells. The most commonly used
disinfectants are sodium hypochlorite, calcium hypochlorite , ethanol and mercuric
chloride (HgCl2).The cultures are incubated in growth chamber either under light or dark
conditions according to the method of propagation.
Stage II: Multiplication stage.
The aim of this phase is to increase the number of propagules. The number of
propagules is multiplied by repeated subcultures until the desired (or planned) number
of plants is attained.
Stage III: Rooting stage.
The rooting stage may occur simultaneously in the same culture media used for
multiplication of the explants. However, in some cases it is necessary to change media,
including nutritional modification and growth regulator composition to induce rooting and
the development of strong root growth.
Stage IV: Acclimatization Stage.

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At this stage, the in vitro plants are weaned and hardened. Hardening is done
gradually from high to low humidity and from low light intensity to high light intensity.
The plants are then transferred to an appropriate substrate (sand, peat, compost etc.)
and gradually hardened under greenhouse.
IMPORTANCE OF TISSUE CULTURE:
 To conserve rare or endangered plant species.
 The commercial production of plants used as potting, landscape and florist
subjects, which uses meristem and shoot culture to produce large numbers of
identical individuals.
 A plant breeder may use tissue culture to screen cells rather than plants for
advantageous characters, e.g. herbicides resistance/tolerance.
 Large scale growth of plant cells in liquid culture in bioreactors for production of
valuable compounds, like plant-derived secondary metabolites and recombinant
proteins used as biopharmaceuticals.
 To cross distantly related species by protoplast fusion and regeneration of the
novel hybrid.
 To rapidly study the molecular basis for physiological, biochemical and
reproductive mechanisms in plants, for example in vitro selection for stress
tolerant plants.
 To cross pollinate distantly related species and then tissue culture the resulting
embryo, this would otherwise normally die.
 For chromosomes doubling and induction of polyploidy, for examples doubled
haploid, tetraploids and other forms of polyploids. This is usually achieved by
application of antimitotic agents such as colchicine or oryzalin.
 As a tissue for transformation, followed by either short term testing of genetics
constructs or regeneration of transgenic plants.
 Production of identical sterile hybrid species can be obtained.
 Removal of viruses by propagation from meristem tissues.
 Only a small explant is enough to get millions of plant with extremely high
multiplication rate.
Micropropagation:
Micropropagation starts with the selection of plant tissues (explant) from a
healthy, vigorous mother plant .Any part of the plant (leaf, apical meristem, bud and
root) can be used as explant.
Embryogenesis:
The process of initiation and development of embryos or embryo-like structures from
somatic cells (Somatic embryogenesis).
Organogenesis:

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The process of initiation and development of a structure that shows natural organ form
and/or function.
Factors determining success with plants TC.
Status of donor plant.
•Species, genotype, age of tissue, explant size and type.
Experimental conditions.
•Temperature, light, day/night.
Composition of culture medium.
Important:
All factors are genotype-dependent and require optimization for each cultivar.

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PATHWAY OF TISSUE CULTURE
Morphogenesis:
 Morphogenesis is the biological process that causes an organism to develop its
shape. Morphogenesis can take place also in a mature organism, in cell culture
or inside tumor cell masses.
↓ ↓
PATHWAY OF MORPHOGENESIS
Direct Morphogenesis
Rooting Under in-Vitro
Plant
Regeneration of Plants From
Callus
Explant
Indirect Morphogenesis
Explant
Shoots
Rooting Under in-Vitro
Plant
Hardening
Plantlets
Hardening
Plant

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CULTURE MEDIA
A growth medium or culture media is a semi solid designed to support the growth of
microorganisms or sell or small plants. There are different types of medium for growing
different types of cells or plant.
Types of media:
Synthetic media :
When a medium is composed of chemically defined components, it is referred to as
synthetic media.
Natural media:
If a medium contains chemically undefined compounds ( e.g. vegetable extract, fruit
juice, plant extract), it is regarded as a natural media.
Commonly used media:-
White’s medium:
This is one of the earliest plant culture media developed for root culture.
MS medium :
Murashige and skoog (MS) originally formulated a medium to induce organogenesis,
and regeneration of plants in cultured tissue. These days, MS medium is widely used for
many types of culture system.
B5 Medium :
Developed by Gamborg, B5 medium was originally designed for cell suspension and
callus culture. Presently used for protoplast culture.
N6 Medium:
Chu formulated this medium and it is used for cereal anther culture, beside other tissue
culture.
Nitsch’s Medium:
This medium was developed by Nitsch and Nitchs and frequently used for anther
culture.
Culture Medium constituents:
• Inorganic salt formulations.
• Source of carbohydrate.
• Vitamins.
• Water.
• Plant hormones - auxins, cytokinins, GA‘3.

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• Solidifying agents.
Plant tissue culture media:
Inorganic compounds (mineral nutrition)
Carbohydrates (typically sucrose)
Plant Growth Regulators (PGRs) (hormones)
Miscellaneous compounds
Plant tissues cultured in vitro require a balanced supply of nutrients.
Essential Nutrients:
Inorganic compounds.
Macronutrients.
•N, P, K, Mg, Ca, S.
Micronutrients.
•Mn, I, Cu, Co, B, Mo, Fe, Zn, (Ni, Al).
Mineral salt composition:
Macroelements: The elements required in concentration > 0.5 mmol/l.
The essential macroelements: N, K, P, Ca, S, Mg, Cl.
Microelements: The elements required in conc. < 0.5 mmol/l.
The essential microelements: Fe, Mn, B, Cu, Zn, I, Mo, Co.
The optimum concentration → maximum growth rate.

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Mineral salts:
Function of nutrients in plant growth:
Plant growth regulators:
(Body building Plants)
Auxins:
- induces cell division, cell elongation, swelling of tissues, formation of callus, formation
of adventitious roots.
- inhibits adventitious and axillary shoot formation
- 2,4-D,

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-NAA,
-IAA,
-IBA,
-pCPA
Cytokinins:
- shoot induction, cell division.
- BAP,
-Kinetin,
-Zeatin,
-2iP
Gibberellins:
Plant regeneration, elongation of internodes
- GA3…
Abscisic acid:
Induction of embryogenesis
- ABA
Cultured tissue must contain competent cells or cells capable of regaining competence
(dedifferentiation). e.g. an excised piece of differentiated tissue or organ (Explant)
→dedifferentiation → callus (heterogenous) → redifferentiation (whole plant) = cellular
totipotency.
1957 Skoog and Miller demonstrated that two hormones affect explants’
differentiation:
– Auxin: Stimulates root development
– Cytokinin: Stimulates shoot development
• Generally, the ratio of these two hormones can determine
plant development:
– ↑ Auxin ↓Cytokinin = Root development
– ↑ Cytokinin ↓Auxin = Shoot development
– Auxin = Cytokinin = Callus development
Typical plant growth regulators and their effects on tissue cultures:

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Auxins →
•callus
•roots
Cytokinins → •shoots
•embryoids
Gibberellic acids →
•cell growth
•elongation
Auxins:
Plant growth and physiological functions.
Phototropism .
Apical dominance.
Cell division.
Differentiation .
Initiation of embryos, organs (esp. roots).
Synthetic auxins are often more effective than the natural auxins.
Cytokinins:
Main effects in tissue culture systems.
Adventitious shoot formation (at high conc.).
Inhibition of root formation.
Cell division.
Callus formation and growth.
Stimulation and outgrowth of axillary buds.
Inhibition of leaf senescence.
Gibberellic acid (GA):
Synthesized irom mevalonate .
Shoot and root apices, embryos, cotyledons, fruit, tubers .
Only some forms are biologically active: GA3.
Dramatic effects on cell elongation.
Promotes cell division in combination with IAA.
Effects on seed germination (breaking seed dormancy).

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Improves fruit set, fruit growth, fruit maturation and fruit ripening.
Promotes flowering.
Solidifying Agents:
For the preparation of semisolid or solid tissue culture media, solidifying or gelling
agents are required. In fact, solidifying agent extend support to tissue growing in the
static conditions.
 Agar: A polysaccharide obtained from seaweeds, is most commonly used as a
gelling agent for the following reasons:
(a) It does not react with media constituents.
(b) It is not digested by plant enzymes and is stable at culture temperature. Agar at a
concentration of 0.5 to 1% in the medium can form a gel.
(c)
 Gelatin: It is used at high concentration (10%) with a limited success. This is
mainly because gelatin melts at low temperature (25%) and consequently the
gelling property is lost.
Other Gelling Agents: Bio-gel (polyacrylamide pellets), Phytagel, gelrite an d
purified agrose are other solidifying agents, although less frequently used.it is in fact
advantageous to use synthetic gelling compounds, since they can form gels at a
relatively low concentration (0.1 to 2.5 gl-1
)
pH of Medium: the optimal pH for most tissue cultures is in range of 0.5-0.6. At a pH
higher than 7.0 and lower than 4.5, the plant cells stop growing in culture. If general, pH
above 6.0 give the medium hard appearance, while below 5.0 does not allow gelling of
the medium.
Nutrient medium pH range of 5.7 to 5.8 is suitable in-vitro growth of explant.

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INSTRUMENTATION:
Following instruments were used in SFRI during study:
a. Types of Autoclave:
1. Horizontal autoclave.
2. Vertical autoclave.
1. Horizontal autoclave:
 It was used for discarding contaminated cultures. Contaminated culture bottles,
test tubes etc. were kept one hour at 15 PSI pressure and 121o
C temperature in
autoclave.
Fig:Horizontal autoclave
2. Vertical autoclave:
 Used for sterilization of media and glassware‘s. Media was kept for 20
minutes and glassware‘s was kept 1 hour at 15 PSI and 121o
C temperature
for purpose of sterilization.
Fig: Vertical autoclave

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b. RO Water Purifier:
 The distillation process removes minerals and can reduce levels of chemical
contaminations.
 A water distiller works by boiling water into water vapour, condensing it and
returning it to its liquid state. It is collection in a storage container.
Fig: RO Water Purifier
c. Microwave oven :
 A Microwave oven was used to melt microbiological media, resulting in a
substantial reduction of heat generation and considerable saving time.
Fig: Microwave oven

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d. PH Meter and Magnetic Stirrer:
 They are used for maintaining the pH of the solution and media. pH of the
media should b in the range of 5.7 to 5.8, which is adjusted by adding 1N HCL
or NaOH.
 Magnetic stirrer was used to stir and mix solutions for precise periods, from
short as a minute to long as a few days.
Fig: pH Meter and Magnetic Stirrer
e. Electronic Balance:
 It is used to weight accurate amount of salts and chemicals for media
preparation.
Fig:Electronic Balance

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f. Hot Air Oven:
 It is used for sterilized of glassware‘s at 60 o
C for one day and 180 o
C for one
hour. Its temperature ranges from 25 o
C to 300 o
C.
Fig: Hot Air Oven
g. Laminar Air Flow:
 They are used for inoculation purpose. Before inoculation LAF cabinet was
wiped with 70% alchol and then exposed to UV Light for 40 minutes.
Fig:Laminar Air Flow

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At the early stages of planning it is important to ensure that the basic infrastructure such
as continuity of power and supply of appropriate quantity of water are in place that local
back-up support service are available for the repair and servicing of equipment‘s. a
standard plant tissue culture laboratory should have facility for:
 Washing and storage of glassware, plastic ware and lab ware.
 Preparation, sterilization and storage of nutrient media.
 Aseptic manipulation of plant material.
 Maintenance of culture under controlled condition of light and temperature.
 Greenhouse for hardening of plantlets.
 HPLC unit for separation of sample.
a. Washing room:
 Should be provided with appropriate washing are, running tap water, brushes
of various shapes and sizes, racks.
 It may also have a dust proof cabinet to store them.
Fig: Washing room
b. Sterilization room:
 Should have hot air oven for drying glassware/ lab wares and sterilization of
lab equipment‘s.
 It should also have autoclave for sterilization of glassware‘s, distilled water and
media.
Fig: Sterilization room
Lab infrastructure

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c. Media preparation room:
 Should have shelves for various chemicals required, refrigerator to store
chemicals, Electronic balance and benches for working.
 Also provided with hot-plate cum magnetic stirrer, pH meter and microware
Oven.
Fig: Media preparation room
d. Inoculation room:
 It should be provided with LAF (Laminar Air Flow) for aseptic manipulation of
explants
 It have table for keeping media, sterilized cotton. Air conditioner and SDDW
(sterilized double distilled water)
Fig: Inoculation room

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e. Culture room:
 It should be provided with controlled condition of light (1000 lux) and air
conditioners to maintain temperature around 25 to -2o
C.
Fig: Culture room
f. Mist chamber :
 It should be provided with proper green shed.
 Also provided with proper water provision and only fertilized soil is used for
Hardening of plant.
Fig: Mist chamber

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GLASSWARE’S AND PLASTIC WARE WASHING
A detergent (Labolene) especially designed for washing are available. Washing is done
in fallowing steps:-
 Overnight soaking of glassware‘s or labwares in detergent solution.
 Thoroughly rinsing first with tap water and then with distilled water.
 Drying in hot air oven at 70_75o
for 1 hour.
 Storing in dusting proof cupboards.
Sterilization Techniques:
 Media, glassware‘s or plastic wares, instruments and plant material (explant)
are sterilized to keep them free from microbes i.e. to maintain aseptic
condition.
 The instructions or equipment‘s used for aseptic manipulation such as forceps,
scissors, scalpels, needles and spatula are normally sterilized by dipping in
95% ethanol followed by flaming and cooling. This is done at the start of the
transfer or inoculation work and several times during operation to minimize the
contamination.
Techniques:
S.No Sterilization techniques Material sterilized
1 Physical method.
Moist heat (autoclaving) (121o
C at 15
PSI for 20 to 40 minutes).
Media, culture vessels glassware‘s
or plastic wares and Contamined
cultures.
2 Dry heat (160o
C to 180o
C for 1 hours). Empty glassware‘s like culture
bottles, pipettes, measuring cylinder,
test tubes etc.
3 Flaming (red hot). Needle. Scalpels, Forceps and
Scissors.

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SCIENTIFIC CLASSIFICATION
Kingdom: Plantae
Order: Poales
Family: Poaceae
Genus: Bambusa
Species: Bambusa tulda
Bambusa tulda Roxb.
About the species

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Bhaans (Kashmir); Jati/Mirtinga/Wat (Arunachal Pradesh); Jati (Assam); Owati
(Meghalaya); Koraincho bans (Sikkim); Longmeii (Ao –Nagaland); Rawthing (Mizoram);
Mirtinga (Tripura).
Habitat:
This species occurs at an altitude of 1500 m. it prefers moist alluvial soil in good rainfall
areas and fine textured soil in semi evergreen forest, in relatively low rainfall areas with
subtropical to temperate climatic condition.
Distribution:
Distributed widely in North Eastern India and West Bengal.
Flowering and fruiting:
This species flower gregariously. The flowering cycle is 30 – to 60 years.
Identification features:
A large tufted bamboo.
Culm up to 20m high and 8 cm in diameter, smooth; internodes 40-70 cm long.
Culm sheath 20-25 cm long and broad, nearly glabrous, rounded at tip, black inside;
blade 10-15 cm long, triangular, cuspidate, appressed hairy beneath, rounded at base;
ligule 2 mm high, white hairy outside.
Leaves 20-35 cm long and 3-4 cm broad, oblong-lanceolate, base oblique, petiole
short; leaf-sheath glabrous or sparsely hairy, ligule short.
Silvicultural management techniques:
The seeds exhibit orthodox behavior and can be stored by proper control of moisture
content and temperature. Studies on seed viability shows that under natural condition
the seed are viable not more than two months but this can be extended by storing over
anhydrous silica gel in desiccators up to 18 months.
Vegetative propagation like rhizome and culm cutting are successfully practiced for
propagation of this species apart from seeds. The seedlings raised from culm cuttings
can be successfully multiplied by shoot proliferation. As per felling rules, felling cycle
Local name

35. SFRI
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four years is suggested. Culm less than one year should be retained and cuttings
should be made 30 cm above the ground. A minimum of six culms should be left in a
clump. This species is one of the high yielding bamboos suggested for large scale
plantation.
Pest and diseases and their control:
The sap sucker Oregma bumbusae which causes the wilting and death of young shoots
have been reported. Bavistin, BHC powder or dialdrin or aldrin (0.5 percent solution or
powder) are affective control. Fungal infection also affects the yield and quality of pulp.
The species also affected by blight caused by Sarocladium oryzae. This can be
controlled by cultural practice and application of Dithane M45as soil drench.
Uses:
It is favored for handicraft, paper and structural purpose. It is a strong bamboo; it lends
itself easily to mechanized processing, and is being used for making bamboo boards
and composites.

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REVIEW OF LITERATURE

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REVIEW OF LITERATURE
Pratibha Sharma and K.P Sarma. In vitro auxiliary shoot formation was highest in
Murashige and Skoog basal medium supplemented with 1.0 mg/l 6-Benzyle Adenine
(BA). Subcultures of shoots from clumps were continued several times for maintaining a
stock of mother culture. Clumps of at least 3 shoots were used for root induction in MS
medium with Indole-3- Acetic Acid (IAA), Indole-3- Butyric Acid (IBA) and naphthalene
acetic acid (NAA).Response of rooting was found more in 5.0 mg/l naphthalene acetic
acid. Rooted plantlets were successfully acclimatized in green house for 20 to 25 days
and then were transferred to the natural field condition. The survival rate was recorded
100 percent in field condition. To the best of our knowledge this is the first report on in
vitro generation of Bambusa tulda from mature field grown auxiliary bud in commercial
scale in North-East India. In this paper, a continuous mass multiplication protocol of B.
tulda was described, which is cost effective, easy to raise, economic to adopt, easy to
transport for selling purpose
In B.tulda, Banik (1987b) was able to increase the seed longevity period up to 18
months by storing over silica gel in a desiccator. Efforts to prolong the viability of fleshy
recalcitrant bamboo seeds by conventional storage methods were not promising.
Reports show that storing the orthodox seeds of bamboos over calcium chloride with a
moisture content of 10-11 % is ideal. The viability of seeds of B. bambos and B. tulda
was extended by storing the seeds over calcium chloride at room temperature.
Seedlings grow well in partial shade compared to direct sunlight. The germinating
plumules are very thin in B. tulda and thick in M.baccifera. Within 1-4 weeks, plumules
elongate rapidly into stems bearing single leaves arising alternately. The stems of B.
tulda, B.longispathus, and B. polymorpha are more or less woody in nature,but M.
baccifera has a soft and succulent stem with vigorous growth. Arhizome system starts
to develop in the seedling one or two monthsafter germination.
Reported that 5-9 month old seedlings of B. tulda can be multiplied 3-5 times in number
through this technique. Every year the seedling can be multiplied at the same rate,
keeping a stock for future macro proliferation. The survival rate of these multiplied
seedlings is 90-100%.
Macro-proliferation, a method of plant multiplication by separating the rooted tillers has
been used by many workers for enhancing the rate of multiplication of in vitro raised
plants and for continuous supply of plantlets. Splitting of rooted tillers could double the
production of Dendrocalamus asper plants (Singh et al., 2011) while three-fold increase
was achieved in and Bambusa balcooa (Mudoi and Borthakur 2009), .B tulda (Mishra et
al., 2011)

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Mishra Y, Patel PK, Yadav S, Shirin F, Ansari SA (2008). A micropropagation system
for cloning of Bambusa tulda Roxb. Sci. Hortic. 115:315-318.
Yogeshwar Mishra, Pradeep Kumar Patel, Suman Yadav, Fatima Shirin, S.A.
Ansari.
The communication describes standardization of an efficient in vitro propagation and
hardening procedure for obtaining plantlets from field grown culms of Bambusa tulda.
Administration for 10 min of 0.05 and 0.1% mercuric chloride to explants collected in
winter and summer seasons, respectively facilitated optimum culture establishment and
bud break. 0.1–0.2% mercuric chloride in rainy season enhanced aseptic culture
establishment but inhibited bud break due to toxicity to explants. MS liquid medium
enriched with 100 mMglutamine, 0.1 mMindole-3-acetic acid and 12 mM 6-
benzylaminopurine supported maximum in vitro shoot multiplication rate of two-fold. The
proliferated shoots were successfully rooted on MS liquid medium supplemented with
40 mM coumarin resulting in a maximum of 98% rooting. The procedure requires 45
days cycle for the in vitro clonal propagation (15 days for shoot multiplication and 30
days for root induction) and 80 days for acclimatized plantlet production.

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METHODOLOGY

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Composition of MS Media
Basal Media:
Compound. Amount. (mg/l)
NH4NO3 1650
KNO3 1900
MgSO4. 7H2O 370
CaCl2.2H2O 440
KH2PO4 170
KI 0.83
H3BO3 6.2
MnSO4.4H2O 22.3
ZnSO4.7H2O 8.6
Na2MoO4.2H2O 0.25
CuSO4.5H2O 0.025
CoCl2.6H2O 0.025
FeSo4.7H2O 27.8
Na2EDTA.2H2O 37.3
vitamins
Inositol 100
Nicotinic acid 0.5
Pyridoxine Hcl 0.5
Thiamine Hcl 0.1
Glycine 2
Stocks Solutions:
Stock-I (20x) Macronutrients.
Compound. Amount (mg/l).
NH4NO3 33000
KNO3 38000
CaCl2.2H2O 8800
MgSO4 7400
KH2PO4 3400
Stock-II (200x) Micronutrients
KI 166
H3BO3 1240
MnSO4.4H2O 4460
ZnSO4.7H2O 1720
Na2MoO4.2H2O 50
CuSO4.5H2O 5

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CoCl2.6H2O 5
Stock-III
(200x)
Iron
FeSo4.7H2O 5560
Na2EDTA.2H2O 7460
Stock-IV
(200x)
Vitamins
Inositol 20000
Nicotinic acid 100
Pyridoxine Hcl 100
Thiamine 20 mg/l
Glycine 400
Volume of stock 2000 ml 1000 ml 500 ml
Stock I 100 ml 50 ml 25 ml
Stock ii 10 ml 5 ml 2.5 ml
Stock iii 10 ml 5 ml 2.5 ml
Stock iv 10 ml 5 ml 2.5 ml`
PGR (Plant Growth Hormone) As per need.
Sucrose 3% (30 g/l)
pH 5.7 -5.8 using 1 Hcl or NaOH
Agar 0.8% (8 g/l)

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
Take 500 ml DDW in a conical flask
Add 8 gm Agar to the solution
Adjust the pH of the solution 5.7-5.8with the pH meter
Now add required amount of PGR
Add 5 ml of Stock-II and stir
Then 5 ml of Stock-IV and shake well
Add 50 ml of Stock-I and stir
Add 5 ml of Stock-III and stir
Add 30 gm Sucrose and dissolve it
Now pour the media in test tubes or bottles
Keep it in the microwave to melt the Agar
Sterilize them in autoclave at 121oC at 15 PSI pressure for 30 minutes
MS Media Preparation
For 1 Litre Media
Important:
 Auxin dissolved in alcohol.
 Cytokines dissolved in NaOH.

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Explants were collected in bottle early in morning
Explant were cleaned with Alcohol and cut required parts
Cover the top of bottle with muslin cloth
Cover the top of bottle with muslin cloth
Wash the explant with DDW for (3-4) times
Wash the explant with DDW for (3-4) times
Wash the explant with Extren detergent
Now washing with 1% Bavistin (Fungicide) for 10 minutes
Keep with under running tap water for 5 minutes
Again wash the explant with DDW for 3-4 times
COLLECTION OF
EXPLANT & WASHING

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WASHING & INOCULATION OF EXPLANT IN LAMINAR AIR
FLOW
Washing the Explants with SDDW for 3-4 times
Labelled with the name of explant, date of inoculation
Now cover the test tubes / bottles along with cap by ceiling tape
Now innoculate the Explant in front of flame and cover the cap tightly
Put the Explants in petriplates and allow to dry
Again wash the Explants with SDDW for 3-4 times
Now wash the Explants with 0.1% (100 mg) HgCl2 for 2 minutes
Finally put the culture bottles or test tubes in culture room under the
proper light and temperature

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↓
↓
↓
↓
↓
PREPARATION AND WASHING OF LAF
Wipe out LAF with 70% alcohol
Put all the requirements and bottles of SDDW and a switch on UV light for
1 HOUR
Expose the LAF Cabinet under UV light for 1 HOUR
Again wipe out LAF, test tubes/bottles containing media with Alcohol
Dip scalpel and forceps into alcohol containing tubes
Wipe out hands with 70% Alcohol

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↓
↓
↓
↓
↓
↓
↓
TECHNIQUE FOR SUBCULTURING
Wipe out LAF 70% alcohol and expose the LAF cabinet with UV light
for 30 minutes
Now put all the requirements including media bottles and fresh
culture tubes and expose to UV for 30 minutes
Wipe out hands and LAF surface with 70% alcohol before use
Forcep, scalpel and petriplates were wiped out with alcohol and flame
sterilized
Put the explants in petriplates and slowly remove the dry part and
media stick to explant to avoid contamination
Properly cut the lower and upper part of explant to remove dead cells
and carefully inoculation the explant in media bottle
Bottles were labeled with name of explant, date of subculturing and
cover the top of bottle tape
Finally put the culture bottles in culture room under the proper light
and temperature
OBSERVATION OF CULTURE VESSELS:
 Culture tubes / bottles were timely observed for
growth and contamination.
 Contaminated cultures were immediately
removed.

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Results and discussion

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EFFECT OF DIFFERENT TIME INTERVAL AS WELL AS CONCENTRATION
AND TREATMENT DURING FRESH CULTURING:
NAME of EXPLANT - BAMBUSA TULDA
PGR
Combination
TREAMENT
% OF RESPOND SHOOTS CONTAMINATION %
SHOOTS
Avg. data
I
week
II
week
III
week
I
week
II
week
III
week
I week II week
III week
No
Length
(cm)
No
Length
(cm)
No Length
(cm)
CONTROL
RT-5min
93% 86% 13% 13%
2.2
EXTRIN-2gm
5 min
BAVISTIN-
2gm, 15 min
86% Nil 1.3 1.2 2.8
3.1 3.0
Hgcl2-0.1% 4
min
1BAP
1mg/l
RT-5min
EXTRIN-2gm
5 min
BAVISTIN-
2gm 15 min
Hgcl2-0.1% 5
min
100% 100% 90% Nil Nil 10% 1.6 1.5 2.6 3.1 3.2 3.5
RT=Running Water
BAP=benzyl 6-amino purine.
 Maximum number of shoot multiplication and their better respond was observed at the
concentration and combination of 1.0 mg/l Benzyl Amino Purine.
 In control there is a less number of shoot multiplication.
From the above table it is recommended that during fresh culturing the effect of Hgcl2
was found to best with 0.1 Hgcl2 concentration for 5 minutes as compared to 4 minutes.
The species of Bambusa tulda showed best response in the PGR combination of 1.0
BAP 1mg/l. so the subculturing of the explants was under process in the same
combination of PGR.
Observation table

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Images Showing The Growth Of
Explant At Different Time Intervals
Figure 1:-Growth after 7 days in 1 mg/l BAP)
Gr
Figure 1:-Growth after 14 days in 1 mg/l BAP)

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Figure 3 Growth after 21 days in (1 mg/l BAP)
.

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Conclusion

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Conclusion
The present study was conducted at the plant tissue laboratory, deparment of Forest
Genetic, Plant Propagation & Biotechnology Division,state Forest Research Institute
Jabalpur Madhya Pradesh, during the period from 30 may to 28 june to propagate from
combinations of the control and PGR medium for micropopagation of Bumbusa tulda.
Murashing and skoog medium was used.
The nodal cutting explants, maximum number of shoot multiplication and their better
respond was observed at the concentration and combination of 1.0 mg/ Benzyl Amino
Purine optimum 3.2 shoot with 3.5 cm length after 21 IIIrd week.+

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Our experiences in SFRI Jabalpur Madhya Pradesh
About wildlife
Mr. R.Bisen (ACF)
Wildlife:
Wildlife is all non-domesticated plants, animals, and other living things.
Domestication, act of taming, or controlling, wild plant and animal species
and producing them for human benefit, is performed often and has an
impact on the environment, both positive and negative.
Wildlife Conservation:
 Wildlife conservation is the practice of protecting endangered plant
and animal species and their habitats.
 Among the goals of wildlife conservation are to ensure that nature
will be around for future generations to enjoy and to recognize the
importance of wildlife and wilderness lands to humans.
 Many nations have government agencies dedicated to wildlife
conservation, which help to implement policies designed to protect
wildlife. Numerous independent nonprofit organizations also
promote various wildlife conservation causes.
 Wildlife conservation has become an increasingly important
practice due to the negative effects of human activity on wildlife.
 The science of extinction is called dirology.
 An endangered species is defined as a population of a living being
that is at the danger of becoming extinct because of several
reasons.
 Either they are few in number or are threatened by the varying
environmental or predation parameters.
Major threats to wildlife
 Habitat loss—due to destruction
 Habitat fragmentation
 Habitat degradation: pollution, invasive species
 Climate change

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 Unregulated Hunting and poaching
 Over exploitation
Wildlife values.
Positive values
Tangible Intangible
Negative values:
 Wildlife damage.
 Man-animals conflict.
 Economic loss.
 Physical utility
 Economic value
 Recreational value
 Historical value
 Scientific value
 Ecological value
 Existence value

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Positive values Tangible:
 Physical utility: as food, clothing and others as domestic
 Economic value: furs, hides, every and medicines
 Recreational value: NP, sanctuary, bird watching, tourism
 Historical value: About historical knowledge of wildlife.
Intangible
Scientific value: research and development of new things.
 Ecological value: maintaining ecological balance.
 Existence value: future potentiality that helps in preservation of
genetic diversity.
Negative values
 Wildlife damage: damage to agriculture as well as forest crops.
 Man-animals conflict: human death, injury and illness and disease
chikungunea.
 Economic loss: reduced the productivity of forest and agriculture
crop.
PRINCIPLES OF CONSERVATION
The management of biosphere to yield sustainable benefit to present
generations, maintaining its potential to meet the needs and aspirations of
future generations‖ is called as conservation. there is an urgent need to
conserve the biodiversity. The conservation of biodiversity has the following
objectives:
 To preserve the biodiversity.
 Maintenance of ecological balance.
 Sustainable utilization of resources for all.
METHODS OF CONSERVATION
In situ conservation: ―Conservation of species in its natural habitat or
ecosystem. It involves setting large areas for wildlife conservation and

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protection of endangered species. There are different categories of
protected areas. They are
1> National Parks,
2> Sanctuaries,
3> Biosphere Reserves,
4> Protected forests
1. National Parks
A relatively large area which is strictly reserved for the conservation of wild
life forms. Deforestation, grazing, cultivation and private ownership are not
permitted in this area. These are designated to conserve specific wild
animals like Tiger, Lion etc. along with other life forms. The parks are
around 100-1000sq.km. and boundaries are circumscribed by legislation.
Except buffer zone, no biotic interference and tourism permissible. There
are 90 national parks are occur in India and some of them are listed
below.
SV National Park – Andhra Pradesh – Leopard, Elephant etc.
Gir NP – Madhya Pradesh – Lions, Antilopes
Khaziranga National Park – Assam – Rhinoceros, Leopard etc.
Sundarbans – West Bengal - Tigers
2. Sanctuaries.
These are meant for conservation of specific animal and plant. The area is
about 100-1000sq.km and there is no legislative boundaries. Grazing,
timber harvesting, collection of NTFP (Non Timber Forest Products) and
private ownership are accepted. There are 492 wildlife sanctuaries in India.
Rajeev Gandhi wildlife sanctuary of Andhra Pradesh is meant for
conservation of Tiger, Leopard and Crocodiles. Madhumalai wildlife
sanctuary (Tamil Nadu) is meant for conservation of Elephant, Four horned
Antelopes, Kumbhalgarh wildlife sanctuary in Rajasmand district Rajasthan
for bears, Sitamata wildlife sanctuary for flying squirrel, Bheinsroadgarh
wildlife sanctuary for leopard.
3. Biosphere reserves
These are meant for protecting and conservation of entire ecosystem.
First time UNESCO (United Nations Education Social & Cultural
Organization) proposed Biosphere reserves in 1972 through Man &

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Biosphere Program . Local tribal people would be allowed to continue
their traditions but have no specific legal status. Commercial activities
and construction of projects not allowed in Biosphere reserves. It
have Core zone, Natural disturbed area, Manipulation zone. . In India
18 Biosphere Reserves are occurred,
4. Protected forests
A protected forest is a forest with some amount of legal or constitutional
protection, or where the habitat and resident species are legally accorded
protection from further deplication .

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Forest mensuration
Mr. SK Jain (Acf)
Introduction: It is an essential part of mathematics. The word is derived
from latin word ‗mensura‘ which means ‗measure‗.
Mensuration is a branch of forestry which deals with the determination of
dimensions (e.g. diameter, height, volume etc), form, age and increment of
single tree, stands or whole woods, either standing or after felling
.(Chaturvedi and Khanna, 1982)
Objectives
 Basis for sale.
 Basis of management.
 Measurement for research.
 Measurement for planning.
Scope
 Provides foundations of measurement principles applicable to any
forest measurement problems.
 Application of statistical theory and use of computer for data
processing
 Forecast of future yields.
 Measurement not only standing trees but also felled timber and
their conversion.
Unit of measurement in forest measurement
 1 mile=1.609km.
 1 cft=0.0283 cu m.
 1 cft/acre= 0.070 cu m/ha.
 1 ha= 2.47105 acres.
 1 cu m/ha= 14.291 cft/acre.
 1 cubic metre= 35.3147 cubic ft.
 1 kilogram= 2.20462 pounds.
 1 metric=0.98420 ton.

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Measurement of Trees
The main objective of measurement of individual trees is to estimate the
quantity of timber, firewood or any other forest produce which can be
obtained from them. It covers:
 Diameter and Girth Measurement.
 Height Measurement.
 Measurement of Logs and Fuel wood.
Diameter Measurement of Trees
 The linear measurement, the main objective of which is to estimate
the volume of the trees.
 The volume of a tree is dependent on diameter or girth at breast-
height, total height and form factor.
 It is not only necessary for calculation of volume of logs, but also
necessary for making inventory of growing stock as well as to
correlate height, volume, age, increment of trees.
DBH Measurement
 DBH is simply the average stem diameter outside bark at point,
1.37m above ground.
 Universally adopted standard height for measuring girth, diameters
and basal area of standing trees India, Burma, America, Union of
South Africa and other British Colonies-1.37m.
 In Europe, U.K., DBH is taken as 1.3m. It is recommended by FAO
as standard.
Significance of DBH
 Convenient height for taking measurement.

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 Avoids the fatigue caused unnecessarily.
 Saves extra expenditure from not clearing the base.
 Abnormalities, eg. Root swell, disappear below breast-height.
 Standardizes diameter measurement giving a uniform point of
measurement. Diameter measurement at stump height is preferred,
but standardization is lost because height of stump depends upon
skill of the labor and the commercial value of the tree.
Points to be considered at the time of measuring diameter
 Breast height point should be marked by intersecting vertical and
horizontal lines 12 cm long, painted with white paint.
 Breast height should be marked by means of a measuring stick on
standing 1.37m or 4 ft 6 in above the ground level, but 1.3 (4‘3‖) in
case of FAO.
 On the sloppy ground, the diameter at breast height should be
measured on the uphill side, after removing any dead leaves or
needles lodged there.
 In the case of leaning tree, dbh is measured alone the tree stem
and not vertically.
 Breast height mark should be shifted up or down as little as
possible to a more normal position of the stem and then diameter
measured if stem is abnormal.
 Buttress is formed due to edaphic factor so if buttress is seen, the
dbh should be taken a little above the buttress formed.
Instruments for DBH measurement
 Mostly used to measure dbh/girth in developing countries are,
 Wooden scale.
 Callipers
 Tape.
 Biltmore stick.
 Penta prism.
 Their use depends upon the condition of trees (felled or standing)
and the degree of accuracy required (research, business, etc.).

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Instruments for Height Measurement
1. Based on Similar Triangles.
 Christen Hypsometer.
 Smythies‘ Hypsometer.
Disadvantages
 It‘s very slow, fatigue, heavy and rough information.
Advantages
 Able to make manually even in the field
Used by unskilled labor.
2. Based on Trigonometrical Principles
 Abney‘s Level.
 Brandis Hypsometer.
 Relaskop.
 Haga Altimeter.
Disadvantages
 Manufactured scientifically, repair is difficult on spot, Limited use
and expensive.
 Only used by skilled labor.
Advantages
 It‘s fast, easy to carry and accuracy maintained available in
markets
 Adopted by many countries.

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Reference

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STATE FOREST RESEARCH INSTITUTE JABALPUR

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SFRI NURSERY
Department of Forest Genetic, Plant Propagation & Biotechnology Division

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Faculty of forestry
Name:ManzoorNabiWani
Father:GhulamNabiWani
DOB:26/10/1995
Address:NewColonyTullamullaGanderbalKmr
CorrespondingAddress:OldHostelRoomNo,20MewarUniversity
Rajasthan
Phone:09982826606/08713861955
EmailId:Manzoornabi57@Gmail.Com
Enrollment no: mur1301982
The forest
Make your heart gentle.
You became one with it.
No place for greed or anger there
Manzoor nabi