TOPIC: - MICROPROPAGATION

SUBJECT:-CONCEPTS OF BIOTECHNOLOGY

SUBJECT CODE: - BTC012

SUBMITTED TO: - SUBMITTED BY:-

MR.HARSH INDESH ATTRI

(LECT. IN BIOTECH) ROLL NO.R108A22

REG.NO.1040070147

B.TECH. BIOTECH

3RD SEM

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 PREFACE

The purpose of this term paper is to understand the process of micropropagation which
helps us to create many organisms which are clones of each other generaly plants tissue
culture is known as microprapgation. This introductory text contains adequate material to
understand the process and the advantages of the micropropagation. This term paper help
us to organize our knowledge into something that can be comprehended in a relatively
short time and still convey a reasonable complete picture of the process of
micropropagation. The primary goal has been to provide useful information in a simple
and illustrative language. The organization of the material is largely traditional and
ordered.Since what is loosely known as biotechnology covers such a wide and diverse
field, I have included terms from biology, biochemistry, and chemistry. In seeking terms
and definitions I have borrowed freely from books, journals, industry periodicals and my
friends.This term paper contains the defination of micropropagation, process of
micropropagation and the advantages and disadvantages of micropropagation.such type
of material is been introduced in this term paper.
I hope that this brief text will enable a better understanding of the complex ideas and
techniques presented in today's biotech work environment.I offer this work in good faith
and in the hope that you should enjoy it reading.

CONTENT

 Introduction

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  Micropropagation

 Methods and procedure

1) Multiplication
2) Pretransplant
3) Transfer from culture

 Plant tissue culture

 Benefits of plant tissue culture

 Advantages and disadvantages of micropropagation

ACKNOWLEDGEMENT

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I would like to express my gratitude to all those who gave me the possibility to complete
this term paper. I want to thank the Department of BIOTECHNOLOGY of LOVELY
PROFESSIONAL UNIVERSITY for giving me permission to commence this Term
paper, to do the necessary research work and to use departmental data. I have furthermore
to thank to Mr.Harsh, Lect.in concepts in biotechnology, who gave and confirmed this
permission and encouraged me to go ahead with my term paper.

I am deeply indebted to my supervisor Lect. Harsh whose help, stimulating suggestions

and encouragement helped me in all the time of research for and writing of this term
paper.

• Thank you

INTRODUCTION

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Micropropagation is the practice of rapidly multiplying stock plant material to produce a
large number of progeny plants, using modern plant tissue culture methods.
Micropropagation begins with the selection of plant material to be propagated. Clean
stock materials that are free of viruses and fungi are important in the production of the
healthiest plants. Often plants are first virus indexed to determine if they are clean and
free of viruses. Once the plant material is chosen for culture, the collection of explant(s)
begins and is dependent on the type of tissue to be used; including stem tips, anthers,
petals, pollen and others plant tissues. The explant material is then surface sterilized,
usually in multiple courses of bleach and alcohol washes and finally rinsed in sterilized
water. Multiplication is the taking of tissue samples produced during the first stage and
increasing their number. Following the successful introduction and growth of plant tissue,
the establishment stage is followed by multiplication. Through repeated cycles of this
process, a single explant sample may be increased from one to hundreds or thousands of
plants. Depending on the type of tissue grown, multiplication can involve different
methods and media. Species that have regeneration problems, especially because of poor
seed set or germination (as in Anogeissus and bamboo). In these cases, seeds collected
from superior trees are used for initiating cultures. Micropropagation will be crucial to
the agricuture industry in the future because it is used to produce plants which have been
genetically modified and selected for their ability to resist certain indigenous
environmental stresses. Micropropagation is not always the perfect means of multiplying
plants .The major limitation in the use of Micropropagatgion for many plants is the cost
of production; for many plants the use of seeds, which are normally disease free and
produced in good numbers, readily produce plants in good numbers at a lower cost.

MICROPROPAGATION

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Micropropagation is the practice of rapidly multiplying stock plant material to produce a
large number of progeny plants, using modern plant tissue culture methods.

Micropropagation is used to multiply novel plants, such as those that have been
genetically modified or bred through conventional plant breeding methods. It is also used
to provide a sufficient number of plantlets for planting from a stock plant which does not
produce seeds, or does not respond well to vegetative reproduction.

Methods

In vitro culture of plants in a controlled, sterile

environment

Micropropagation begins with the selection of plant

material to be propagated. Clean stock materials that
are free of viruses and fungi are important in the
production of the healthiest plants. Often plants are first
virus indexed to determine if they are clean and free of
viruses. Once the plant material is chosen for culture,
the collection of explant(s) begins and is dependent on
the type of tissue to be used; including stem tips,
anthers, petals, pollen and others plant tissues. The explant material is then surface
sterilized, usually in multiple courses of bleach and alcohol washes and finally rinsed in
sterilized water. This small portion of plant tissue, sometimes only a single cell, is placed
on a growth medium, typically containing sucrose as an energy source and one or more
plant growth regulators (plant hormones). Usually the medium is thickened with agar to
create a gel which supports the explant during growth. Some plants are easily grown on
simple media but others require more complicated media for successful grow; some

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media include vitamins, minerals and amino acids. The medium is sterilized during
preparation to prevent fungal and bacterial contamination, which can outgrow and
smother the growing explant. Autoclaves and filter sterilization are used to remove
potential contaminates, under smaller scales of production a pressure cooker is often
used.

The plant tissue grows and differentiates into new tissues depending on the medium. For
example, media containing cytokinins are used to create branched shoots from plant buds.

Multiplication

Multiplication is the taking of tissue samples produced during the first stage and
increasing their number. Following the successful introduction and growth of plant tissue,
the establishment stage is followed by multiplication. Through repeated cycles of this
process, a single explant sample may be increased from one to hundreds or thousands of
plants. Depending on the type of tissue grown, multiplication can involve different
methods and media. If the plant material grown is callus tissue, it can be placed in a
blender and cut into smaller pieces and recultured on the same type of culture medium to
grow more callus tissue. If the tissue is grown as small plants called plantlets, hormones
are often added that cause the plantlets to produce many small offshoots that can be
removed and recultured.

Pretransplant

This stage involves treating the plantlets/shoots produced to encourage root growth and
"hardening." It is performed in vitro, or in a sterile "test tube" environment.

Root growth does not always occur in the earlier stages in plant cell culture, and is of
course a requirement for successful plant growth after the micropropagation procedure. It
is often performed in vitro by transferring the plantlets to a growth medium containing
auxin(s) which stimulate root initiation. The pretransplant stage is not always performed;

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Some plants are micropropagated and grown in culture and normal cuttings are made that
are then rooted ex vitro.

"Hardening" refers to the preparation of the plants for a natural growth environment.
Until this stage, the plantlets have been grown in "ideal" conditions, designed to
encourage rapid growth. Due to lack of necessity, the plants are likely to be highly
susceptible to disease and often do not have fully functional dermal coverings and will be
inefficient in their use of water and energy. In vitro conditions are high in humidity and
plants grown under these condition do not form a working cuticle and stomata that keep
the plant from drying out, when taken out of culture the plantlets need time to adjust to
more natural environmental conditions. Hardening typically involves slowly weaning the
plantlets from a high-humidity, low light, warm environment to what would be
considered a normal growth environment for the species in question. This is done by
moving the plants to a location high in humidity, such as a green house with regular mist
watering.

Transfer from culture

In the final stage of plant micropropagation, the plantlets are removed from the plant
media and transferred to soil or (more commonly) potting compost for continued growth
by conventional methods.

This stage is often combined with the "pretransplant" stage.

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CRITERIA FOR PLANT TISSUE CULTURE-:
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.

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Figure 1 - Areas of Tissue Culture Collection

The standard protocol for performing plant tissue culture experiments is fairly basic.
First, it is essential that a sterile environment be created. The medium used to grow the
plant tissue, the plant tissues themselves, and the environment surrounding the tissue

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culture, must be free of all possible contaminants. The presence of any bacterial, fungal,
algal, or viral contaminants could potentially rob the desired plants of the nutrients
provided by the culture medium and have devastating effects upon their growth Once a
sterile environment has been established, tissue can be collected from the plant’s leaf,
shoot, bud, stem, or root. Because each of these cells is totipotent, each has the potential
to express an entire organism. The tissue sample can then be placed on an aseptic (free of
microorganisms), nutrient-rich medium where its cells will begin to grow and develop
into the desired plant product (The nature of the medium and the nutrients that it contains
is dependent upon the type of plant being grown and the properties that the grower
wishes to express. Finally, the developing tissue should be maintained in a closely
controlled chemical and physical environment, such as a greenhouse, to achieve the best
results

Figure 2 - The Basic Steps of Micropropagation

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The benefits of plant tissue culture are extensive in the agricultural world.
Micropropagation is favorable to traditional crop breeding methods in many respects, the
first being that it allows for the production of huge numbers of plants in a very short
period of time. In the Netherlands alone, over 100,000,000 plants are produced using
micropropagation each year .Plant tissue culture is also advantageous to growers because
the overwhelming number of plants can be produced using the tissue collected from a
single parent plant – a plant which itself remains unharmed in the tissue harvesting
process. Crop production through micropropagation also eliminates the possibility of any
interruption in the growing season because it can be carried out inside the carefully
regulated environment of a greenhouse. Because the chemical and physical environment
inside a greenhouse can be closely monitored, any lull in production that might typically
occur as a result of seasonal change can be avoided.

Species are selected for tissue culture on the following basis:

Species that have regeneration problems, especially because of poor seed set or
germination (as in Anogeissus and bamboo). In these cases, seeds collected from superior
trees are used for initiating cultures.

Species that vary markedly in their desirable traits, i.e. Eucalyptus. The selected trees are
marked from the variant population for the desirable trait such as disease resistance,
straight bole, higher productivity, etc. in consultation with officials from state forest
department or growers

Species where plants of any one particular sex is of commercial importance, for example
female plants of papaya and male plants of asparagus

Micropropagation will be crucial to the agricuture industry in the future because it is used
to produce plants which have been genetically modified and selected for their ability to
resist certain indigenous environmental stresses. Currently, scientists and members of the
agricultural community have joined forces to investigate the possibility of creating lines
of tomatoes that possess increased salt tolerance (to be grown in areas in which the soil is

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high in salinity), plants that are completely resistant to various viral, bacterial, algal, and
fungal infections, tobacco plants whose leaves can withstand freezing temperatures, and
crops that are entirely resistant to harmful and destructive insects.

Advantages:-

Micropropagation has a number of advantages over traditional plant propagation

techniques:

• The main advantage of micropropagation is the production of many plants that are
clones of each other.
• Micropropagation can be used to produces disease-free plants.
• Micropropagation produces rooted plantlets ready for growth, saving time for the
grower when seeds or cuttings are slow to establish or grow.
• It can have an extraordinarily high fecundity rate, producing thousands of
propagules while conventional techniques might only produce a fraction of this a
number.
• It is the only viable method of regenerating genetically modified cells or cells
after protoplast fusion.
• It is useful in multiplying plants which produce seeds in uneconomical amounts,
or when plants are sterile and do not produce viable seeds or when seed can't be
stored (vgr. recalcitrant seeds).
• Micropropagation often produces more robust plants, leading to accelerated
growth compared to similar plants produced by conventional methods - like seeds
or cuttings.
• Some plants, including most orchids, can only be grown from seed using
micropropagation techniques.

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. 1 The chief advantage of micropropagation is the extremely high multiplication rates,
e.g., 106 plants/year from single explants.

2. Very small explants can be used for micropropagation, which is impossible with
conventional technique.
3. During micropropagation, fungi and bacteria are usually eliminated so that the plants
obtained are clean, while conventional methods propagate the disease as well.

4. Plants can be maintained in vitro in a pathogen-free state. Such plants are easy to
export since there is no quarantine problem, and their packing is easier due to their
smaller size.

5. In dioecious species (male and female plants), plants of one sex may be more desirable
than those of the other, e.g., male Asparagus or female papaya plants. In such cases,
plants of the desired sex can be selectively multiplied by micropropagation.

6. In case of forest trees, mature elite trees can be identified and rapidly cloned by this
technique.
7. Micropropagation can be carried out throughout the year independent of seasons.
8. In case of many ornamentals, tissue culture plants give better growth, more flowers
and less fall out.

Disadvantages:-

Micropropagation is not always the perfect means of multiplying plants, conditions that
limit its use include:

• It is very expensive, and can have a labor cost of more than 70%
• An infected plant sample can produce infected progeny. This is uncommon if the
stock plants are carefully screened and vetted to prevent culturing plants infected
with virus or fungus.

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 • Not all plants can be successfully tissue cultured, often because the proper
medium for growth is not known or the plants produce secondary metabolic
chemicals that stunt or kill the explant.
• Sometimes plants or cultivars do not come true to type after being tissue cultured,
this is often dependent on the type of explant material utilized during the initiation
phase or the result of the age of the cell or propagule line.
• Some plants are very difficult to disinfest of fungal organisms.

The major limitation in the use of Micropropagatgion for many plants is the cost of
production; for many plants the use of seeds, which are normally disease free and
produced in good numbers, readily produce plants in good numbers at a lower cost. For
this reason, many plant breeders do not utilize micropropagation because the cost is
prohibitive, other breeders use it to produce stock plants that are then used for seed
multiplication.

Mechanisation of the process could reduce labour costs, but has proven difficult to
achive, despite active attempts to develop technological solutions.

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References

• TEXT BOOKS:-
 1. Biotechnology-expanding horizons: b.d.singh. Kalyani publishers . 2nd
editoin
 2. Molecular biotechnology: s.b.parimrose. Panima publication. 2nd
edition

INTERNET:-

WWW.GOOGLE.COM

1) en.wikipedia.org/wiki/Micropropagation
2) en.wikipedia.org/wiki/Plant_tissue_culture
3) www.molecular-plant-biotechnology.info/plant-biotechnology

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