Block 3 PLANT BIOTECHNOLOGY PDF

BBYET-143
ECONOMIC BOTANY AND

Indira Gandhi
National Open University
PLANT BIOTECHNOLOGY
School of Sciences
Block
3
PLANT BIOTECHNOLOGY
UNIT 10
Introduction to Biotechnology 9
UNIT 11
Plant Tissue Culture (I) 34
UNIT 12
Plant Tissue Culture (II) 56
Course Design Committee
Prof. A.K. Bhatnagar School of Sciences, IGNOU
Department of Botany, University of Delhi
Prof. Sujatha Varma (Director)
Dr. Sneh Chopra
Kalindi College, University of Delhi Prof. M.S. Nathawat (Ex. Director)
Prof. Jaswant Sokhi (Retd.)
Prof. Bano Saidullah (Retd.)
Prof. Pushplata Tripathi (Retd.)
Prof. Neera Kapoor
Prof. Amrita Nigam
Block Preparation Team

Prof. Amrita Nigam Editor
SOS, IGNOU,
New Delhi-110068 Dr. A.K. Kavathekar (Retd.)
Department of Botany,
Dr. Ravi Rajwanshi
Sri Venkateswara College,
SOS, IGNOU,
New Delhi-110068 Dhaula Kuan,
New Delhi-110021
Dr. Bhupinder Dhir
Consultant, SOS, IGNOU,
New Delhi-110068
Course Coordinator : Prof. Amrita Nigam and Dr. Ravi Rajwanshi
Production Team
Mr. Rajiv Girdhar Mr. Hemant Kumar
AR (P), MPDD, IGNOU SO(P), MPDD, IGNOU
Acknowledgements:
• Mr. Manoj Kumar for CRC Preparation.
May, 2022
Indira Gandhi National Open University, 2022
ISBN
All rights reserved. No part of this work may be reproduced in any form, by mimeograph or any other
means, without permission in writing from the copyright holder.
Further information on the Indira Gandhi National Open University courses may be obtained from the
official website of IGNOU at www.ignou.ac.in.
Printed and published on behalf of Indira Gandhi National Open University, New Delhi by Director,
SOS, IGNOU
BLOCK 3 : PLANT BIOTECHNOLOGY
Block 3 deals with plant biotechnology. Three units (units 10 to 12) of this block introduce
you to the concept of biotechnology, plant biotechnology and the techniques involved in it.
You will study about origin, development of technique of biotechnology, scope of plant
biotechnology and its uses. Biotechnology originated long back when animals were
domesticated, plants were cultivated and microbes were used in getting many products.
Later on the concept of biotechnology was applied to various areas such as agriculture and
medicine. You will be studying about plant biotechnology and main techniques used in it. A
brief account about origin of Plant tissue culture and its applications will also be discussed.
Unit 10 of this block introduces you to the concept of Biotechnology, its origin and
development with time. A brief account of major contributions of scientists is also given. You
will appreciate the concept of the classical, modern day biotechnology, major events in the
development of Biotechnology and methods involved used in it. Various methods used in
Plant Biotechnology will also be discussed in this Unit.
Unit 11 of this block introduces you to the concept of plant tissue culture. You will appreciate
a brief historical account related to the development of the plant tissue culture technique.
Detailed information related to various equipments and methods of plant tissue culture is
provided to you. The concepts such as totipotency, organogenesis, embryogenesis and
protoplast culture is also discussed in detail in this unit.
Unit 12 is devoted exclusively to the applications of Plant Tissue Culture. In this unit, you will
be studying in detail about techniques such as micropropagation, production of androgenic
and gynogenic haploids, embryo culture and secondary metabolite production. The role of
Plant tissue culture in germplasm conservation will also be discussed in the last part of the
unit.
Objectives
After studying this block you will be able to :
describe the concept of origin of biotechnology;
list out the applications of biotechnology;
appreciate the role of biotechnology in improvement of plants;
enlist the major techniques and methods in plant biotechnology;
describe the importance of plant tissue culture; and
enumerate the major applications of plant tissue culture.
7
8
Unit 10 Introduction to Biotechnology
UNIT 10
INTRODUCTION TO
BIOTECHNOLOGY
Structure
10.1 Introduction 10.7 Methods in Plant
Biotechnology
Objectives
10.8 Applications of Plant
10.2 General account
Biotechnology
Various Stages in the
Crop Improvement
Development of Biotechnology
Genetic Transformation
10.3 Types of Biotechnology
Industrial
10.4 Various Techniques in
Biotechnology Pharmaceutical
10.5 Applications of 10.9 Summary

Biotechnology
10.10 Terminal questions
10.6 Plant Biotechnology
10.11 Answers
10.1 INTRODUCTION
Humans have been using biological processes to improve the quality of life for
the last 10,000 years. The biological agents such as microorganisms have
been used to get various products. Use of living organisms to develop
technologies and products that help improve our lives and the health is
referred as Biotechnology. The concept of biotechnology began to develop in
the 19th century when plant and animal cells as well their constituents were
cultured in vitro conditions to get desired products. Since then
microorganisms have been used for large-scale production of biochemicals
(such as alcohol, antibiotics), food processing and environmental remediation.
The major applications of biotechnology include therapeutics, diagnostics,
food processing, bioremediation, waste treatment, production of energy and
genetically modified crops for improvement of agriculture.
The advent of Recombinant DNA technology has made development of

microbes, plants and animals that possess superior and novel capabilities 9
Block 3 Plant Biotechnology
possible. Genetically Modified Organisms (GMO) have been created by
transferring one or more genes from one organism to another. Genetically
modified plants have been developed for tolerance to high temperature,
salinity stress and resistance to pathogen and pest attack. These approaches
have proven to be very useful in increasing the yield of crop plants and
reducing post-harvest losses. Development of crop plants with the improved
nutritional value and reduced reliance on chemical pesticides (pest-resistant
crops) has been made possible via technique of Biotechnology. The present
Unit introduces you to the concept of biotechnology and provides you with
information about various methods of plant biotechnology and its applications.
Objectives
Objectives
After studying this unit, you would be able to:
describe the concept of biotechnology;
discuss the basics of plant biotechnology;
enlist various methods in plant biotechnology; and
enumerate various applications of plant biotechnology.
10.2 GENERAL ACCOUNT

Since ancient times, man has been using living things to improve his life. In
the early times (about 8000-4000 B.C.) man has been using plants and
animals for various services. As the time passed, various developments took
place in the area of biotechnology.
Various stages in the development of Biotechnology
The development of biotechnology can be categorized as
• Ancient biotechnology (8000–4000 BC): Early history of biotechnology

relates to domestication of animals and cultivation of plants.
• Classical biotechnology (2000 BC; 1800–1900 AD): This era of

biotechnology was based on use of microbes in fermentation, production
of food and medicine. In this period Genetics was developed (1900–
1953) and DNA research was initiated (1953–1976).
• Modern biotechnology (1977): In this period, genetic information in

organisms was manipulated via genetic engineering.
Ancient biotechnology
In the period before the year 1800, events such as domestication of animals to
get milk or meat, cultivation of plants such as rice, barley and wheat as major
source of food were categorized as biotechnological developments. Breeding
of plants and animals has been done to improve them by introducing desirable
10 characteristics.
Classical biotechnology
The production of cheese, yogurt and bread from micro-organisms, alcoholic

drinks such as beer and wine via fermentation has been done since ancient
times. The role of microorganisms (such as bacteria, yeast or molds) in
hydrolysis sugars and hence fermentation was discovered. In 1796, Edward
Jenner demonstrated role of vaccination in providing immunity against
smallpox, hence is considered the founder of vaccinology. Scientific evidence
for fermentation was first described by Louis Pasteur in the late 1800s. He
proposed a theory known as germ theory stating microorganisms play a major
role in the process of fermentation. Honey, soybean curds has been used as
natural antibiotic and found effective in healing wounds since ancient times.
Ukrainian farmers used moldy cheese to treat infected wounds. Later it was
found that antibiotics present in molds killed bacteria and prevented the
spread of infection. Enzymes and proteins have been discovered in 1830.
In 1919, Hungarian engineer Karl Ereky coined the term “Biotechnology".

According to him, biotechnology is the use of living organisms to produce
materials or products of importance. In other words, it is the technology that
uses biological processes, organisms or systems to produce products that
improve human lives.
In 1920, Chaim Weizman tried to produce useful chemical compounds

through biological techniques. He used Clostridium acetobutylicum that
converted starch to butanol. Later, biotechnology such as fermentation was
refined to develop paint solvents for the automobile industry and acetone from
starch. These processes were promoted during World War I. During World
War II, penicillin was discovered. In 1929, Alexander Fleming produced
antibiotic penicillin from cultures of Penicillium notatum. The penicillin proved
useful for the treatment of wounded soldiers during World War II (Fig. 10.1).
The focus of biotechnology shifted to pharmaceuticals in 1930s. Also in1930s
use of biotechnology shifted to agriculture sector.
Selective breeding of plants and animals has been done in the past. In this
procedure organisms with desirable traits were allowed to mate to further
enhance these traits in their offspring. It was found that selective breeding
could improve yield as well as productivity. Mule is one of the most primitive
examples of crossbreeding for the benefit of humans. Man started using mules
for transportation, carrying loads and farming. Later on focus of biotechnology
moved to pharmaceuticals. The microorganisms were used in preparation of
antibiotics.
At the end of the eighteenth century and the commencement of nineteenth

century, focus of biotechnology was shifted to crop rotation (including
leguminous crops), development of vaccination and animal based studies. The
complexity of biotechnology increased owing to the evolution of new
technologies and better understanding of various principles of life science.
By the end of the nineteenth century, many microorganisms were discovered,

Mendel's work on genetics was accomplished and microbial processes such
as fermentation were explored. At the beginning of the twentieth century,
industry and agriculture started using biotechnology methods. 11
(a) (b)
Fig. 10.1: a) Karl Ereky; b) Alexander Fleming.
Modern biotechnology
Later in 1960s and 70s molecular, cellular technologies began to emerge. In

the period between1953 to 1976, DNA research was initiated. Cells and
biological molecules (DNA, RNA and proteins) were used to make useful
products. The real biotechnology was initiated with the production of Insulin in
1970. The first restriction enzyme was also discovered in 1970. The discovery
enabled the researchers to insert foreign genes in bacteria. This formed the
basis of recombinant DNA technology and is considered the birth of modern-
day biotechnology. The DNA of living organisms was manipulated and the
products obtained from genetically modified organisms were used in the
manipulation of molecules. In 1977, researchers in Scotland cloned a sheep
named Dolly, later on, sheep named Polly was cloned using nuclear transfer
technology as it had human gene encoding Factor IX, a protein involved in
preventing haemophilia. Companies such as Genentech, Amgen, Biogen,
Cetus, and Genex started were established in the mid-to late 1970s. They
manufactured genetically engineered substances primarily for medical and
environmental uses. In 1998, the importance of cloning Dolly was realized.
Molecular biologists became interested in studying stem cells. In 1999,
antibody analysis was found as another tool of use. A glance of the historical
events in biotechnology has been provided in Table 10.1.
Table 10.1: A summary of historical events in Biotechnology.
Peiod Time/Year Event

Pre-1800 6000 BC Yeast was used to prepare beer
4000 BC In Egypt, yeast was used to prepare bread.
420 BC Greek philosopher Socrates found that similar
characteristics are found between parents and their
offspring.
320 BC Greek philosopher Aristotle gave the concept that
characters get inherited from father.
1000 AD Theory of abiogenesis, or spontaneous generation
was proposed.
12
1630 William Harvey discovered the circulation of blood in
the body.
1660–1675 Marcello Malpighi found blood circulation in
capillaries using a microscope and found that the
brain is connected to the spinal cord by bundles of
fibers which form the nervous system.
1673 Antonie van Leeuwenhoek identified micro-
organisms such as protozoa and bacteria, and
suggested that micro-organisms play an active role
in fermentation.
1701 Giacomo Pylarini found that the administration of
smallpox prevents its occurrence later in life. The
procedure was termed 'vaccination'.
1800–1900 1809 Nicolas Appert invent a technique using heat to
sterilize food
1856 Justus von Liebig, Pasteur (1822–1895) suggested
that microbes are responsible for fermentation.
1859 Charles Darwin (1809–1882) speculated 'natural
selection'.
1863 The method of pasteurization was discovered by
Pasteur.
Heinrich Anton de Bary found that fungus was
responsible for potato blight.
1865 Mendel suggested ‘the laws of heredity’.
1868 Johannes Friedrich Miescher separated nuclein (a
compound made of nucleic acid) from pus cells.
1870 Walther Flemming discovered mitosis.
1871 Koch investigated anthrax and explored techniques
to identify, culture and stain micro-organisms.
1880 Louis Pasteur explored weakened (attenuated)
strains of micro-organisms that might not be
virulent.
1881 Koch explained techniques for harvesting bacterial
colonies on potato slices, gelatin and agar medium.
1884 Gram described the differential staining technique
for cellular peptidoglycan-containing bacteria now
known as Gram staining.
1900–1953 1900 Mendel's work was revived by de Vries, von
Tschermak and Correns.
1902 Sutton found that chromosomes (paired) contain
certain elements which are transferred from one
generation to another.
1905 Edmund Beecher Wilson and Nettie Stevens
demonstrated that a single Y chromosome
determines maleness, while two copies of the X
chromosome decide femaleness.
1905–1908 William Bateson and R C Punnett found that several
genes alter or modify the action of other genes. 13
1906 Paul Erlich also investigated atoxyl compounds and
discovered the important features of Salvarsan (the
first chemotherapeutic agent).
1907 Thomas Hunt Morgan revealed that chromosomes
have a defined role in heredity. He discovered
mutation theory into fruit flies.
1909 Wilhelm Johannsen used the word 'gene' for the
carrier/transporter of heredity. He also coined the
terms 'genotype' and 'phenotype'.
1910 Morgan demonstrated that genes are present on
chromosomes.
1911 Morgan established the separation of certain
inherited features that are generally linked to the
separation/breaking of chromosomes during the
process of cell division.
1912 William Lawrence Bragg discovered the application
of X-rays in the determination of the molecular
structure of crystalline substances
(Crystallography).
1918 Herbert M Evans found that human genetic material
is made up of 48 chromosomes.
1926 Hermann Joseph Muller discovered that X-rays are
responsible for genetic mutations in fruit flies.
1928 Frederick Griffiths observed the 'transforming
principle' by which a rough type of bacterium is
transformed to a smooth type.
Alexander Fleming studied an old culture of bacteria
infected by fungus Penicliium notatum and
discovered antibiotic penicillin
1938 Proteins and DNA were studied by means of X-rays.
The term 'molecular biology' was coined.
1941 George Wells Beadle and Edward L Tatum
proposed 'one gene, one enzyme' theory.
1943 Canadian scientist Oswald Theordore Avery
discovered that DNA is the carrier of genes.
1943-1953 Cortisone, a steroid hormone was considered as the
first biotech product.
1944 Selman Abraham Waksman explored streptomycin,
an antibiotic active against TB.
1945-1950 Animal cell cultures were harvested for the first time
in laboratories, giving birth to the field of animal
tissue culture.
1947 Barbara McClintock first demonstrated
'transposable elements' known as 'jumping genes'
with the capability to move (or jump) from one site
on the genome to another site.
1950 Erwin Chargaff discovered that the same levels of
adenine and thymine are present in DNA, as are the
same levels of guanine and cytosine. These
associations were later named 'Chargaff's rules'.
14
1952 George Otto Gey created a cell line known as HeLa
from a human cervical carcinoma.
1953–1976 1953 Watson and Crick's article based on unfolding the
double-helix structure of DNA was published in the
journal, Nature.
1953–1976 The discovery of the structure of DNA revolutionized

molecular biology and genetics.
1957 Crick and Gamov studied 'central
dogma',demonstrating how DNA functions to
construct protein.
1958 Arthur Kornberg created DNA in a test tube for the
first time. The mechanical protein sequencer, the
Moore–Stein amino acid analyzer, was developed.
1959 François Jacob and Jacques Lucien Monod
documented concept of genetic regulation. They
explained the concept of 'repressor' and 'operon'.
1960 A French researcher discovered messenger RNA
(mRNA).
1961 François Jacob and Jacques Monad gave the
concept of Operon.
1962 Watson and Crick were awarded the Nobel Prize in
Physiology or Medicine with Maurice Wilkins.
1963 Samuel Katz and John F Enders developed the first
vaccine for measles.
1964 Enzyme reverse transcriptase was discovered.
1966 The genetic code was explored by several
researchers. Marshall Warren Nirenberg, J Heinrich
Matthaei and S Ochoa reported that a genetic
sequence of three nucleotide bases (called codons)
decides each of 20 amino acids.
1967 Arthur Kornberg reported a study using single-
stranded natural viral DNA.
1969 An enzyme is synthesized in vitro conditions.
1970 Virologists Peter H Duesberg and Peter K Vogt
identified the first oncogene in a virus.
Restriction enzymes were discovered.
1967-1971 Maurice Hilleman made the first American vaccine
for mumps. The first vaccine for rubella was
developed.
1972 Paul Berg, a biochemist, utilized a restriction
enzyme to cut DNA into fragments. He employed a
ligase enzyme to join two DNA strands concurrently
to form a hybrid circular molecule.
1973 Bruce Nathan Ames, a biochemist developed an
investigation to distinguish chemicals that damage
DNA. Later, the Ames test became extensively used
to identify cancer-causing substances.
15
Stanley Cohen and Herbert Boyer used bacterial
genes to perform the first successful rDNA
experiment.
Sir Edwin Mellor Southern developed a blotting
technique for DNA called the Southern blot.
1974 The first vaccine for chicken pox was developed.
1975 Colony hybridization and Southern blotting were
explored for identifying specific DNA sequences.
The first monoclonal antibodies were prepared.
1976 J Michael Bishop and Harold Varmus established
the presence of cancer-causing genes called
oncogenes on animal chromosomes.
The NIH published the first guidelines for rDNA
research.
1977– 1977 With the advent of genetic engineering it was
(Modern possible to produce human protein in bacteria for
biotechnology) the first time.
R Austrian and his coworkers developed the first
vaccine for pneumonia.
1978 Herbert W Boyer synthesized synthetic human
insulin by introducing the insulin gene into the
bacterium Escherichia coli.
Louise Brown, the first test-tube baby, was born in
the UK.
The first vaccine for meningococcal meningitis was
developed
1980 Kary Mullis and coworkers established a tool for
multiplying DNA sequences in vitro using the
polymerase chain reaction (PCR).
1981 Baruch Blumberg and Irving Millman developed the
first vaccine for hepatitis B. The first transgenic
animal (mice) was produced.
1982 US Food and Drug Administration (FDA) allowed
the first genetically engineered drug in the form of
human insulin produced by bacteria.
Michael Smith at the University of British Columbia,
Vancouver, established a procedure for producing
precise amino acid changes anywhere in a protein.
1983 The first genetically modified (transgenic) plant is
formed.
1984 The DNA fingerprinting technique was discovered.
The first genetically engineered vaccine was
discovered for hepatitis B.
1985 The NIH published guidelines for performing
experiments in gene therapy on humans.
Genetically engineered plants resistant to viruses,
insects and bacteria were field-tested for the first
time.
16
1986 The NIH sanctioned guidelines for executing trials of
gene therapy on humans.
The automated DNA sequencer was discovered in
California.
1987 Calgene Inc. obtained a patent for the tomato
polygalacturonase DNA sequence, which was later
used to synthesize an antisense RNA sequence that
can further extend the shelf life of fruit.
Maynard Olson and colleagues at Washington
University discovered yeast artificial chromosomes,
which are expression vectors for large proteins.
1989 A gene responsible for cystic fibrosis was explored.
1990 Human Genome Project was launched.
1993 Kary Mullis won the Nobel Prize in Chemistry for
inventing the tool of PCR. FDA approved a
recombinant protein to treat multiple sclerosis.
A global research team, led by Daniel Cohen
created a rough map of all 23 pairs of human
chromosomes.
1994 A number of genes, human and others were
identified and their functions explained.
1995 The first vaccine for hepatitis A was explored.
Researchers at the Institute for Genomic Research
completed the first full gene sequence of a living
organism for the bacterium Haemophilus influenzae.
1997 Researchers at the Roslin Institute in Scotland
cloned a sheep called Dolly from the cell of an adult
ewe. Dolly was the first mammal cloned by a
technique called nuclear transfer technology.
The first human artificial chromosome was
discovered.
1998 A group of researchers succeeded in culturing
embryonic stem cells.
A draft of the human genome map is created that
locates more than 30,000 genes.
Embryonic stem cells were employed to regenerate
tissue and produce disorders mimicking diseases.
2000 Sir Hargobind Khorana synthesized DNA in a test
tube. He demonstrated the role of nucleotides in
protein synthesis and helped in finding the genetic
code.
2001 The journals Science and Nature reported the
human genome sequence.
2002 A very rapid shotgun sequencing of major genomes
was completed
2003 Celera and the NIH successfully finished the
sequencing of the human genome.
17
2004 The FDA supported the first monoclonal antibody for
cancer therapy
2006 The FDA sanctioned a recombinant vaccine against
human papillomavirus.
Researchers established the three-dimensional (3D)
structure of HIV.
2008 Japanese chemists developed the first DNA
molecule made nearly entirely of artificial parts.
2009 Scientists identified three new genes connected with
Alzheimer's disease, paving the way for possible
new diagnostics and therapeutics.
2013 Researchers established the three-dimensional (3D)
structure of HIV, which causes AIDS.
2010 Researchers from the J.Craig Ventere Institute
create the first synthetic cell.
Modern biotechnology, thus has developed as a science with enormous

potential for human welfare in areas ranging from food processing to human
health and environmental protection. Biotechnology is now being used in
numerous disciplines including bioremediation, energy production, agriculture
and food processing. Biotechnology has vast scope in agriculture for the
production of plants that are resistant to insects, weeds and plant diseases.
Production of medicines (biopharmaceuticals) through cloning of vectors with
genes of interest (GOIs) has been made possible via biotechnology. Modern
biotechnology is applied in medicine and healthcare in therapeutics, mainly for
the discovery, development and production of novel drugs and in diagnostics,
for protein and nucleic acids based tests. In the area of environment,
biotechnology has been primarily used to treat waste and prevent pollution.
Thus modern biotechnology makes significant contribution in improving the
lives of humans.
10.3 TYPES OF BIOTECHNOLOGY

In the recent years, the scope of biotechnology has expanded. It has been
categorized into different types depending on the field in which it is used.
1. Medical Biotechnology
It refers to use of living cells and other materials to improve health of humans.
It is used for finding cure for diseases or prevention of diseases, as a research
tool for studying human health, understanding pathogens and human cell
biology. The technique is used for producing pharmaceutical drugs as well as
other chemicals to combat diseases. The field is used for development of new
drugs and treatments. Examples- vaccines, an anti-lymphoma vaccine has
been developed using genetically engineered tobacco plants that exhibit RNA
(a similar chemical to DNA) from malignant (actively cancerous) B-cells.
2. Agricultural Biotechnology
Biotechnology has been used in developing genetically modified plants to

18 increase crop yields or introduce characteristics that impart tolerance against
biotic, abiotic stress, and provide resistance against pest and pathogen attack.
Examples- development of crops that express anti-pest characteristics. The
genes of Bacillus thuringiensis have been transferred to crops. A protein (Bt)
produced by Bacillus thuringiensisis found to be very effective against pests
such as the European corn borer.
3. Industrial Biotechnology
Biotechnology also finds its applications in industrial sector. It includes use of

microorganisms, or components of cells like enzymes, to generate products
such as food and feed, chemicals, detergents, paper and pulp,
textiles, biofuels, and biogas that are industrially useful. Examples-
Biocatalysts have been developed via industrial biotechnology to synthesize
chemicals.
4. Environmental Biotechnology
Biotechnology is used in treatment of waste and prevention of pollution. These

methods efficiently clean up wastes and significantly reduce our dependence
on methods for land-based disposal. Examples- in Bioremediation, potential of
living organisms are exploited for removal/treatment of wastes. Enzyme
bioreactors that pretreat industrial and food waste components and allow their
efficient removal via sewage system has been developed.
Recently a new system of classifying Biotechnology has been adopted. In this

type of classification, various colours have been given to various branches of
biotechnology (Table 10.2, Fig. 10.2).
Table 10.2: Categorization of various branches of Biotechnology on the

basis of colour.
Color designation Description

Gold biotechnology or Use of computational techniques for analysis of
Bioinformatics or biological data.
computational biology
Red relates to medicine and veterinary products. It
Biotechnology (Biopharma) includes development of new drugs, vaccines and
antibiotics, regenerative therapies, molecular
diagnostics and genetic engineering techniques to
cure diseases applying genetic manipulation.
White Biotechnology includes designing of energy-efficient, less polluting,
and low resource-consuming processes and
products.
Yellow Biotechnology relates to the use of biotechnology in food production
such as making wine, cheese, and beer by
fermentation.
Grey Biotechnology Includes environmental applications of biotechnology
such as removal of pollutants or contaminants using
microorganisms and plants.
Green Biotechnology Use of biotechnology in agriculture for creating new
plant varieties of agricultural interest, biopesticides,
19
and biofertilizers. This area includes production of
transgenics via genetic modification.
Blue Biotechnology use of marine resources to create products and their
application in various sectors.
Violet Biotechnology deals with the law, ethical and philosophical issues
related to biotechnology
Dark Biotechnology Relates to bioterrorism, biological weapons and
biowarfare using microorganisms, and toxins to
cause diseases and death in humans, domestic
animals, and crops.
Fig. 10.2: Categorization of branches of Biotechnology on the basis of colour.
10.4 VARIOUS TECHNIQUES IN

BIOTECHNOLOGY
Various techniques have been followed in biotechnology. Some of the major
ones include:
• Genetic Engineering Technology
The technique involves the manipulation of the DNA of an organism by

inserting foreign DNA.
• Protein Engineering Technology
The technique involves improvement in existing proteins or creating novel

proteins to make useful products.
20 • Enzyme engineering
This technology involves improvement in efficiency of enzyme or formulation
of an enzyme activity by altering its amino acid sequence. Genetic
engineering techniques are widely used to improve enzyme efficiency.
• Cell and Tissue Culture Technology
In this technique, cells/tissues are grown under laboratory conditions to

produce a organism or new products.
• Antisense or RNAi Technology
RNA interference (RNAi), is a mechanism that involves use of small RNAs

(less than 30 bases long) in regulating the expression of genes in eukaryotic
organisms (Fig. 10.3). An RNAi-based mechanism has found its applications
in studies of gene function and therapeutics for the treatment of disease.
Fig. 10.3: An overview of RNAi technique.
• Bioinformatics Technology
Computational analysis of biological data, e.g., sequence analysis

macromolecular structures, high-throughput profiling data analysis.
• Protein separation and identification techniques
Techniques such as electrophoresis, microarrays and blotting techniques that

are helpful in separation and identification of desired DNA.
• Genomics
The genome approaches are used to determine the biological function of

genes and their products. Transcriptomics (profiling of microarray expression),
proteomics (structures/modifications/interactions of proteins), metabolomics
(e.g. metabolite profiling, chemical fingerprinting, flux analysis) are some of its
diversifications (Fig. 10.4). 21
Fig. 10.4: Genome approaches used to determine the biological function of

genes and their products.
10.5 APPLICATIONS OF BIOTECHNOLOGY

Biotechnology has been successfully applied in various areas such as the
environment, medicine, agriculture, industries and many more.
1. Environment
Biotechnological techniques have been used in the diagnosis of

environmental problems, their monitoring and abatement. This technology has
been used in areas of waste management, bioremediation (use of living
organisms to treat environmental pollutants), phytoremediation (use of plants
to remove pollutants from the environment). Apart from these renewable
resources, biodegradable products and alternative energy sources have also
been developed using biotechnology. Biotechnology is used to engineer
microbes that show immense potential for environmental clean-up by
removing a wide range of pollutants from various sectors of the environment.
2. Medicine
Biotechnological techniques have been used in the diagnostics, therapeutics,

vaccines, human Genome Research. The development of insulin and vaccines
such as hepatitis B are some of the major application of biotechnology in
medicine.
Recombinant DNA technology has made an immense impact in the area of

healthcare by enabling the mass production of effective therapeutics.
Transgenic animals are been used to understand how genes contribute to the
development of human diseases such as cancer, cystic fibrosis, rheumatoid
arthritis and Alzheimer’s. Gene therapy had made the treatment of hereditary
diseases possible via the insertion of genes into individual’s cells and tissues.
Box 10.1: Role of biotechnology in abatement of Covid 19 pandemic.
Biotechnology has emerged as a leader in the fight against COVID -19. Vaccines for
the treatment of corona virus infection have been developed using biotechnological
tools. Pfizer-BioNTech and Moderna developed high efficacy mRNA vaccines against
COVID-19. The mRNA vaccine developed by BioNTech/Pfizer was shown to be 90%
effective in preventing COVID-19 infection. Other vaccines are also been developed
22 based on other technologies such as an interference RNA that blocks the virus in the
form of nebulizers, recombinant ACE2 proteins that trick the virus into not binding to
human cells, and antibodies that attack the virus.
Viral vector vaccines, AZD1222 and Sputnik V were developed by AstraZeneca.

mRNA vaccines, mRNA-1273 and BNT162b2, were developed by Moderna
(Cambridge, MA, USA) and Pfizer (New York, USA)
3. Agriculture
Biotechnological techniques have proved very useful in the area of developing

crops with better nutrition content, high yield, and resistance to various abiotic
and biotic stresses. Food, horticultural crops and tree species have been
modified successfully using genetic engineering. For e.g. Oil seed can be
modified to produce fatty acids for petrochemicals, fuels. Efforts have been
made to develop vaccines from plants. Crops that show tolerance against a
broad range of pests/insects have been developed.
4. Industries
Biotechnology has been applied for the production of cellular structures or

production of biological elements for numerous uses such as development of
new materials in the construction industry, and the manufacture of beer and
wine, washing detergents, and personal care products.
Biotechnology is used in the textile industry for the finishing of fabrics and
garments. It helps in production of warmer, stronger, wrinkle and shrink-
resistant clothes along with other properties such as improved dye uptake and
retention, enhanced absorbency. Biotechnology has been successfully applied
in the food processing industry.
5. Biofuels
Biotechnology has been used in sector of energy production. Bioremediation

waste can be converted to biofuel to run generators. Bacteria that degrade
sulfur liquor, which is a waste product of the paper manufacturing industry, can
help in production of methane.
6. Evolutionary studies
Genes associated with ecological traits and evolutionary diversification can

easily be traced using biotechnological tools.
Other Uses
Microbes can be induced to produce enzymes required to turn plant and

vegetable materials into building blocks for biodegradable plastics.
Table 10.3: Applications of biotechnology in different areas.
Area Applications
Plant Transgenic plants, production of secondary metabolite,
biotechnology production of pathogen-free plants or crop improvement,
production of herbicide-resistant crops, pest-resistant ('Bt
concept' pest-resistant transgenic) plants, drought resistance,
flood resistance, salt tolerance, high-yielding GM crops, nitrogen
23
fixing ability, acidity and salinity tolerance, in vitro germplasm
conservation, genetic variability, in vitro pollination, induction of
haploidy, somatic hybridization, genetic transformation,
molecular pharming, somatic embryogenesis, organogenesis,
phytoremediation, in vitro plant germplasm conservation, mutant
selection, somaclonal variation, plant genome analysis, hybrid
seeds, artificial seeds.
Animal Biopharmaceuticals: Production of hormones, growth factors,
biotechnology interferons, enzymes, recombinant proteins, vaccines, blood
components, oligonucleotides, transcription factor-based drugs,
oligonucleotides.
Antibiotics, Diagnostics: antibodies, biosensors, PCR,
therapeutics, vaccines, medical research tools, human genome
research, development of biosensors
Gene therapy, Stem cell therapy, Animal tissue culture: Cell,
tissue and organ culture, Gene cloning: rDNA technology,
genetic engineering, transgenic animals, antibiotics, DNA
markers, animal husbandry, xenotransplantation, medical
biotechnology,
Biopharmaceuticals (drug or vaccine developed through
biotechnology), therapeutants, i.e. products used to maintain
health or prevent disease, biopharming, i.e. production of
pharmaceuticals in cultured organisms,
Biopolymers, Designer drugs
Agricultural crop biotechnology, horticultural biotechnology, tree
biotechnology biotechnology, food processing, plant biotechnology
(photosynthesis improvers, bio-fertilizers, stress-resistant crops
and plants, bio-insecticides and biopesticides)
Food: Increased milk production
Pharmaceuticals: Animals engineered to produce human
proteins for drugs, including insulin and vaccines
Environmental Environmental monitoring, Waste management, Pollution
biotechnology prevention.
Fuel and fodder renewable fuels, Tissue culture technique for rapid afforestation
of degraded forests and regeneration of green cover
Industrial Metabolite production (acetone, butanol, alcohol, antibiotics,
biotechnology enzymes, vitamins, organic acids), anaerobic digestion (for
methane production), waste treatment (both organic and
industrial), production of bio-control agents, fermentation of food
products, bio-based fuel and energy, recovery of metals and
minerals, bioethanol, pulp and paper, food, textiles and leather,
pharmaceuticals, an enzymatic process for producing
antibiotics.
Aquatic Aquaculture, restoring and protecting marine ecosystems,
biotechnology improving seafood quality, environmental remediation, marine
byproducts for human health, biomaterial and bioprocessing,
marine molecular biotechnology.
10.6 PLANT BIOTECHNOLOGY

Plant biotechnology has gained importance because of its role in manipulating
plants for improved agronomic performance. Plant biotechnology involves
24 processes or scientific techniques that are used to develop beneficial plants
and/or their products and development of new plant traits. The use of
biotechnological tools helps in achieving crop improvement at a faster rate and
facilitates the transfer of genes from unrelated species.
The two processes involved in plant biotechnology are tissue culture and
genetic engineering. Plant tissue culture is the most popular technique of plant
biotechnology. The plant tissue culture had its beginning in 1939 when
Gautheret tried to cultivate isolated cells and root tips on an organized
medium. The tissue culture techniques provide the platform for the rapid
multiplication of plant species. Plant tissue culture technique has been used
for getting regeneration of plant cells for endangered/extinct plant species,
rapid production of elite varieties with superior genotypes on a large scale in a
comparatively short time. The plants are genetically modified plants by
transferring genes from one organism to plant to get the desired
characteristics. Plants have been genetically modified to induce herbicide
tolerance, salinity tolerance, drought tolerance, pest resistance, enhanced
nitrogen-fixing ability, improved nutritional value and food quality.
Plant biotechnology also provided an option for germplasm conservation and

production of disease-free varieties. Increased production of biochemical
constituents (secondary metabolites) in plants has also been achieved using
this technique. Production of artificial seeds, biopharmaceuticals, plant-made
pharmaceuticals, therapeutic proteins and plant-made vaccines or antibodies
(plantibodies) is the other area where plant biotechnology is being applied.
SAQ 1
a) Answer in one word only:
i) Who coined the term ‘biotechnology’?
ii) What is the ‘part’ of plant used for culture called?
iii) Name the scientist who tried to cultivate isolated cells and root tips
on an organized medium.
iv) Name the antibiotic produced by Alexander Fleming.
v) Name of the sheep cloned by researchers in Scotland.
vi) Name the technique used for computational analysis of biological

data.
b) Match the following:
Column A Column B
i) Yellow 1. environmental applications of biotechnology

biotechnology
ii) 1984 2. Arthur Kornberg created DNA in a test tube
for the first time.
iii) 1990 3. Har Gobind Khorana synthesized DNA in a

test tube
iv) Grey 4. Sheep cloned using nuclear transfer

biotechnology technology 25
v) 1958 5. Human genome project was launched
vi) Dolly 6. The DNA fingerprinting technique was

discovered.
vii) 2000 7. use of biotechnology in food production
10.7 METHODS IN PLANT BIOTECHNOLOGY

Various methods in plant biotechnology include:
• Genetic engineering/ recombinant DNA technology
• Tissue culture
• Molecular Assisted Breeding
Genetic engineering
Alteration of genetic material (hereditary material) in an organism forms the
basis of genetic engineering. Genetic engineering or recombinant DNA
technology involves the removal of the gene(s) from one organism and
transfer to another organism. Integration of new DNA into a plant’s original
DNA creates a transgenic plant or genetically modified organism (GMO). The
basic requirements for successful genetic engineering are restriction enzymes,
cloning vehicles (vectors) to carry the genes of interest and detection and
selection of cloned genes.
Gene transfer techniques used in genetic engineering include:
• Agrobacterium-mediated gene transfer: The desired trait is isolated from

the DNA of an organism, inserted into Agrobacterium and the target
plant is infected. Cells that accept the DNA are grown into plants with the
new trait.
• Gene gun: DNA that codes for the desired trait is coated onto tiny
particles of tungsten and fired into a group of plant cells. Cells that
accept the DNA show the desired trait.
• Chemicals such as polyethylene glycol (PEG) help in rapid uptake of

DNA by plant protoplasts and integration into the plant’s chromosomal
DNA.
• Electroporation is another method in which short high-voltage electrical

pulses induce the formation of transient micropores in cell membranes
allowing DNA to enter the cell and then the nucleus.
• Apart from these lipofection, microinjection, calcium phosphate

transfection are some other methods that are also followed to transfer
DNA.
Agrobacterium vectors are commonly used for the transfer of transgenes into
plants. The DNA sequences may be cleaved at several points and the
resulting fragments inserted into different parts of the genome (Fig. 10.5).
Transgenes inserted into the genome contain regulatory elements and a
26 selectable marker, often an antibiotic‐ or herbicide‐resistant gene.
Fig. 10.5: An overview of plant genetic engineering.

Plant tissue culture
Cells, anthers, pollen grains, or other tissues are cultured under laboratory
conditions to raise the plants. They are then multiplied in large numbers.
Under cultural conditions, parts of plants (explants) or tissues form callus
(undifferentiated mass of cells) develop directly into embryos (somatic
embryos). The plant tissue culture allows breeders to introduce new cultivars
at a much faster rate due to rapid multiplication of plants.
The tissue culture process involves placing of tissue on a nutrient medium,

multiplication of cells to form callus or the embryos followed by their transfer to
medium containing plant growth regulators and finally root formation (Fig.
10.6). The plantlets are transferred to greenhouses. A large variety of
ornamental plants, crops and medicinal plants have been raised via plant
tissue culture technique.
Cell culture helps in producing clones of a single cell, embryo-rescue,

introgression of characters such as disease resistance into elite breeding lines
and production of haploid plants for developing homozygous breeding lines.
This technique is now widely used for the improvement of important crops
including major cereals, legumes and trees.
Fig. 10.6: Diagrammatic representation of the plant tissue culture technique.
Molecular Assisted Breeding (MAB)/Marker Assisted Selection (MAS)

Molecular breeding is the technique of using DNA markers that are tightly
linked to phenotypic traits to assist in selection. Genetic markers such as 27
microsatellites, random amplified polymorphic DNA (RAPD), restriction
fragment length polymorphisms (RFLPs), amplified fragment length
polymorphism (AFLP) are important components of molecular breeding.
Molecular markers have been developed for many crops including trees. The
information generated from molecular markers, transcriptome profiling, and
genome sequencing facilitate genetic improvement in plants. The markers can
be used to track the presence of valuable characters in large segregating
populations as part of a crop-breeding program. For example, if a useful trait
such as disease resistance or high oil yield is linked with a specific marker,
many hundreds or even thousands of young plantlets can be screened for the
likely presence of the trait without growing the plants to maturity. The use of
molecular markers can decrease the time scale of crop-breeding programs by
several years.
Genomic selection is emerging as a molecular breeding method used for crop

improvement. It assists in the identification of superior genotypes with higher
breeding values and helps in segregating breeding populations (Fig. 10.7). It is
based on genome-wide marker profile data. MAS have proved as an efficient
breeding method for drought and salt-tolerant oilseed crops such as Brassica.
It has provided valuable contributions in the designing and development of
oilseed crop ideotype(s).
28 Fig. 10.7: Process of marker assisted breeding technique.

SAQ 2
Fill in the blanks with appropriate words:
a) Improvement of crops done via incorporation of desired agronomic traits

is known as …………………. .
b) The plants formed after incorporation of genes for desired characters are
called ………………….. .
c) Undifferentiated mass of cells produced via tissue culture is called

…………… ..
d) During ……………….. phase of the plant tissue culture, callus or the

embryos are transferred to a medium containing plant growth regulators.
e) Polymorphic DNA (RAPD), amplified fragment length polymorphism

(AFLP) are examples of ………………. .
10.8 APPLICATIONS OF PLANT

BIOTECHNOLOGY
Plant biotechnology has broad applications in several areas. These include
basic research and commercial applications. The basic application of plant
biotechnology deals with understanding the concepts such as molecular
pathways in plant cells. Plant biotechnology techniques such as tissue culture
and genetic engineering have been applied to plants for modification and
improvement. These include raising of pathogen-free plants, germplasm
conservation, clonal propagation, secondary metabolite production and study
of cell behaviour. Biotechnology has got its application in crop improvement
programs.
Various areas of application of Plant Biotechnology are:

• Improvement of varieties for various agronomic traits such as:
Crop improvement (resistance to biotic stress: pests, viruses,

pathogens, abiotic stress tolerance to drought, salinity)
Value addition: enrichment of crops with vitamins.
Increasing the shelf life to delay the fruit ripening
Food processing
Ornamental plants for characters such as size, color, smell
• Phytoremediation: removal of contaminants
• Biofuels: bioenergy crops
• Commercial products such as biopolymers, therapeutic proteins,

biodegradable plastics, etc.
• protection of natural biodiversity 29

10.8.1 Crop Improvement
Plant tissue culture technique
Tissue culture helps in getting rapid clonal multiplication/propagation of plants.
This is called micropropagation. The progeny obtained by vegetative
propagation i.e. asexual reproduction of a single plant or individual is called a
clone. Shoot buds produced on explants are separated and rooted to get
plantlets. The technique proves useful in eliminating viruses and other
pathogens.
Plant cell suspension culture and immobilized cells are being used to get
large-scale production of useful chemical compounds. Cell and organ
culture helps in the conservation of endangered genotypes and
preservation of vegetative tissues. The plants that do not produce seeds
(sterile plants) or have ‘recalcitrant’ seeds can be preserved via in vitro
techniques. Cryopreservation involves the storage of cells or tissues in
liquid nitrogen at ultralow temperature (−196°C) under in vitro conditions.
This method helps in the long-term conservation of essential biological
material and genetic resources. The embryonic tissues are generally
cryopreserved for future use.
10.8.2 Genetic Transformation

In the genetic transformation technique, the genes with the desirable trait are
transferred into host plants to get transgenic plants. In plants, genetic
transformation is done by vector-mediated i.e. Agrobacterium-mediated
genetic transformation, or vector less i.e. direct gene transfer method. The
technique offers a great potential for genetic improvement in various crop
plants. The technique involves integration of plant biotechnology and breeding
programmes. Traits such as increased yield and better quality can be
introduced into plants. For e.g.: Development of the transgenic ‘golden rice’.
Transgenic rice contains three genes encoding the enzymes responsible for
the conversion of geranylgeranyl diphosphate to β-carotene (provitamin A).
The transgenic rice produced is enrich in iron content. The plants are obtained
by expressing ferritin or metallothionein transgenes, or making the existing
iron more available by reducing levels of the iron-sequestering protein,
phytase.
The use of transgenes can help in producing cells having a high capacity of
the desired compounds that can have industrial uses. The modification of
exogenous compounds by plant cells is called biotransformation. The
bioconversion reactions are catalyzed by the enzymes present within the plant
cell. The genetic engineering method has proven useful in developing plants
that show resistance to or protection against various pests, diseases, viruses.
10.8.3 Industrial
Plants produce fuels, proteins, antibodies (plantibodies) and other products
which are of commercial use. For e.g. avidin and β-glucuronidase (GUS) are
proteins produced in transgenic maize. Avidin is used as a biochemical
reagent for research and diagnostics, and also as a biopesticide. Plant oils are
used as a feedstock for the production of oleochemicals. The soybean crop
30 has been transformed to get high oleic acid content.
Non-toxic biodegradable polymers called polyhydroxyalkanoates (PHAs) are
produced by plants and their production can be increased at the agricultural
level. It is possible to obtain a high yield of the polymer from leaves or seeds.
Attempts are been made to obtain transgenic plants so that production of
these copolymers in the plant can be increased (including one in oil palm).
Starch produced by plants has also got uses in industry. Interest is also
developed in manipulating complex carbohydrates in cereal crops such as
rice, wheat, maize and barley. The starches are used for the development of
packaging materials or even biodegradable plastics. Many of the enzymes
involved in starch biosynthesis have been characterized and their genes
cloned and attempts are been made to develop plants with enhanced capacity
for the production of starch.
10.8.4 Pharmaceuticals
Plant-made pharmaceuticals, secondary metabolites, proteins, drugs and
vaccines have been produced via classical and non-classical techniques such
as plant cell culture and genetic engineering.
Apart from these, a new branch of biotechnology i.e. nanobiotechnology which

involves the use of nanomaterials has proven useful in the development of
crops mainly by manipulating the plant nutrient content, imparting disease and
pesticide resistance in plants. Metallic nanoparticles exhibiting relatively
superior anti-pathogenic, anti-fungal and anti-bacterial qualities are being used
in developing nano pesticides. Nanoparticles of zinc oxide and iron helps in
efficient uptake of nutrients by the plant, hence act as nanofertilizers. Their
success depends on factors like plant species, susceptibility and the size,
composition and chemical properties of nanomaterials.
SAQ 3
a) Define the following:
i) Molecular breeding
ii) Cryopreservation
iii) Electroporation
iv) Nanobiotechnology
b) Explain the term ‘transgenic’? How are they useful?.
10.9 SUMMARY
• Use of biological agents such as microorganisms, plants, cells of higher
organisms and their constituents for generating desired products is
called biotechnology.
• Biotechnology has been used since ancient times in as the animals were
domesticated, crops were cultivated and micro-organisms were used to 31
get products such as cheese, yogurt, bread, beer and wine. Later
antibiotics were discovered. Genetics laid the foundation for modern day
biotechnology.
• Modern biotechnology has got its applications in areas such as

medicine, healthcare, therapeutics, diagnostics for development and
production of novel drugs, agriculture for development of improved crop
varieties, environment for treatment of waste and prevents pollution.
• Plant biotechnology is referred to the use of plant cells, tissues, organs

for the generation of useful products. The plant parts (explants) are
cultured under in vitro conditions to get plantlets. Plant biotechnology
plays a promising role in the field of agriculture, industry and
pharmaceutical sciences.
• Plant biotechnology has many applications such as large scale

production of biochemicals, rapid clonal multiplication, development of
homozygous lines via production of haploids, conservation of germplasm
etc.
• The main methods in plant biotechnology are plant tissue culture,

genetic engineering and marker assisted breeding.
• Clonal multiplication, raising of virus-free plants can be done via plant

tissue culture technique. The development of plants with tolerance
against abiotic and biotic stresses, better nutritional quality, better
storage can be achieved via manipulation of DNA by genetic
engineering.
• The plant tissue culture technique can also be used for getting products
of commercial value. These include bioplastics, biofuels, edible plant-
based vaccines, bio-oils, etc
10.10 TERMINAL QUESTIONS

1. Discuss various techniques of Biotechnology.
2. Describe in brief various applications of Biotechnology.
3. How does Biotechnology be used for the improvement of crop plants?
4. Describe the methods of Plant Biotechnology.
5. Enumerate various applications of Plant Biotechnology.
6. How does Plant Biotechnology play a role in germplasm conservation?
10.11 ANSWERS
Self-Assessment Questions
1. a) i) Karl Ereky in 1919
ii) explants
32 iii) G. Haberlandt
iv) Penicillin
v) Dolly
vi) Bioinformatics
b) i) Use of biotechnology in food production
ii) The DNA fingerprinting technique was discovered
iii) Human genome project was launched
iv) environmental applications of biotechnology
v) Arthur Kornberg created DNA in a test tube for the first time.
vi) Sheep cloned using nuclear transfer technology
vii) Har Gobind Khorana synthesized DNA in a test tube
2. a) transformation
b) transgenic
c) callus
d) multiplication
e) genetic markers
3. a) i) Refer to Section 10.7.
ii) Refer to Subsection 10.8.1.
iii) Refer to Section 10.7.
iv) Refer to Subsection 10.8.4.
b) Refer to Subsection 10.8.2.
Terminal Questions
1. Refer to Section 10.4.
Acknowledgements
Fig. 10.1 : Source:

https://www.google.co.in/imgres?imgurl=https%3A%2F%2Fw
instonchurchill.hillsdale.edu%2Fwp-
content%2Fuploads%2F2018%2F09%2FFleming.jpg
33
UNIT 11
PLANT TISSUE CULTURE (I)
Structure
11.1 Introduction 11.5 Totipotency
Objectives 11.6 Organogenesis
11.2 Historical Account 11.7 Embryogenesis
11.3 Equipment Required for 11.8 Protoplast Isolation, Culture

Plant Tissue Culture and Fusion
Basic Steps in Plant Tissue 11.9 Summary

Culture
11.10 Terminal Questions
11.4 Composition of Media
11.11 Answers
11.1 INTRODUCTION
The process of raising plants under in-vitro aseptic conditions by culturing
explants (parts of differentiated tissues) in a nutrient medium under conditions
is termed as plant tissue culture. Cells, tissues, organs, or whole plant is
cultured under in vitro conditions. Ideal conditions such as the proper supply of
nutrients, pH and temperature provide a favorable environment for the growth
and multiplication of plants under in vitro conditions. Plant tissue culture
technique plays a significant role in the area of crop improvement. The plant
tissue culture technique has been successful in getting large-scale production
of plants in a short time duration. Many economically important plant species
have been improved using the tissue culture technique. In addition, the
technique has also contributed to increasing our understanding related to
factors responsible for growth, metabolism, morphogenesis and differentiation
in plants. Plant propagation, production of disease-free, nutritionally improved
and highly tolerant (to conditions such as salinity drought, cold) plants has
been achieved using plant tissue culture technique. Production of secondary
metabolites of immense importance has been increased via this technique.
Besides, many endangered, threatened and rare species can be successfully
grown and conserved using this technique. Tissue culture techniques have
been used for the improvement and propagation of various types of plants
including cereals, grasses, legumes, vegetable crops, root and tuber crops,
34 oilseeds, fruits, plantation crops, forest trees and ornamentals.
Unit 11 Plant Tissue Culture (I)
Before studying in detail about various practical applications of plant tissue
culture technique, we need to understand the basic methods involved in
culture and the requirements for the implementation of this technique. The first
part of this unit provides you with an overview of various scientists that played
an important role in developing the plant tissue culture technique, the various
steps of the technique and the major requirements (equipments and
chemicals). The second part of this unit describes the important aspects of
concepts of totipotency, techniques such as Organogenesis, Embryogenesis
and Protoplast culture.
Objectives
Objectives
list the contributions of various scientists in the plant tissue culture

technique;
explain the conditions and other requirements for raising plants through
tissue culture technique;
explain the concept of ‘totipotency’ and describe the processes of

‘Organogenesis’ and ‘Embryogenesis’; and
appreciate the techniques of protoplast isolation, culture and fusion.
11.2 HISTORICAL ACCOUNT

The German Botanist Guttlieb Haberlandt was first to propose the importance
of plant tissue and cell culture in 1902. He grew the tissue of Lamium
puroureum and Eichhornia crassipes, the epidermis of Ornithooalium and
epidermal hairs of Pulmonaria mollissima on a salt solution with sucrose. He
was the first to culture single palisade cells from leaves in Knop’s salt solution
enriched with sucrose. He observed growth in the cells. The cells grew in size,
changed shape, accumulated starch and showed thickening of cell walls. The
cells showed no division. The cells remained alive for up to 1 month. This
happened because highly differentiated cells lack the growth hormones
required for inducing division in mature cells. His experiments were
unsuccessful but they laid down the foundation of tissue culture technology.
He is regarded as the ‘father of plant tissue culture’. He predicted that artificial
embryos can be successfully cultivated from vegetative cells. He was the first
one to propose the concept of totipotency.
The first true plant tissue cultures were obtained by Gautheret. He was able to
raise cambial tissue from Acer pseudoplatanus. He was also able to culture
explants of Ulmus campestre, Robinia pseudoacacia, and Salix capraea using
agar-solidified medium of Knop’s solution, glucose and cysteine hydrochloride.
Later, he included indole acetic acid and vitamin B in the medium to get carrot
root tissue cultures and a hybrid of Nicotiana glauca and Nicotiana langsdorffii.
The tissues are grown in a culture medium get differentiated into roots and
shoots. 35
Mature embryos of plants like Raphanus sativus were excised by Hanning
(1940) and successfully grown on mineral salts and sugar solutions. Van
Overbeck (1941) and co-workers demonstrated the stimulatory effect of
coconut milk on embryo development and callus formation in Datura. These
studies proved a turning point in the field of embryo culture as young embryos
could be cultured successfully under in vitro conditions. Braun (1959) was able
to generate a plant from a mature plant cell. The foundation of commercial
plant tissue culture was laid down in 1960 by Morel. He was able to get
multiplication of Cymbidium (an orchid) under culture conditions.
Later Raghavan and Torrey (1963), Norstog (1965) and his coworkers
developed synthetic media for the culture of younger embryos. Laibach (1925,
1929) was able to show the practical application of embryo culture. He was
successful to isolate embryos from seeds of a Linum plant and raise them to
maturity on a nutrient medium. Robbins and Kotte (1922) successfully cultured
isolated root tips of maize, pea, and cotton. In 1934, White reported success in
cultures of tomato root tips. Extensive work on root culture was done by Street
(1957). He cultured excised tomato roots to understand the role of inorganic
ions, carbohydrates, amino acids, hormones and vitamins in plant growth.
Gautheret (1934), White (1939) and Nobecourt successfully cultured cells of
Salix, Nicotiana and carrot on synthetic media. They demonstrated that the
addition of growth regulators and vitamins in media leads to the proliferation of
cells forming a callus.
Skoog (1944), Tsui (1951), and Miller (1955) demonstrated induction of

divisions in isolated, mature, and differentiated cells using syntheticmedia
supplemented with natural compounds. The technique of growing single cells
of Tagetes erecta and Nicotiana tobacum in a liquid medium was developed
by Muir (1953). Vasil and Hildeprandt (1965) raised whole plants from single
cells of tobacco. Skoog and Miller (1957) demonstrated that changes in the
relative concentrations of the two substances (growth hormones) in the
medium regulate the differentiation of organs. The formation of an embryo
from carrot tissue was reported by Reinert and Steward in 1958-59. Whole
plants of Lupinus and Tropaeolum could be raised by culturing shoot tips.
Ball (1946), Morel and Martin (1952) raised virus-free Dahlia plants by
culturing shoots. Murashige used the plant tissue culture technique to multiply
several plants species (ranging from ferns to foliage, flower and fruit plants) in
large numbers. Guha and Maheshwari (1966) demonstrated the possibility of
raising large numbers of haploid plants from pollen grains of Datura. The first
somatic hybrid was produced via protoplast fusion by Carlson and coworkers
in 1972. The use of cell wall degrading enzymes was done in protoplast
culture technology in the 1970s. The totipotency of protoplasts was first
demonstrated by Takebe and his coworkers in tobacco plants. Interspecific
hybrid plants of tobacco were regenerated from mesophyll protoplasts.
With time, various developments were noted in the plant tissue culture
techniques. The major developments have been summarized in Table 11.1.
36
Table 11.1: Developments in Plant tissue culture technique with time.
Year Contribution
1902 Haberlandt proposed the concept of in vitro cell culture.
1904 Hannig cultured embryos from several cruciferous species.
1922 Kolte and Robbins successfully cultured root and stem tips
respectively.
1926 Went discovered the first plant growth hormone i.e. Indole acetic acid.
1934 White introduced vitamin B as a 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.
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 a higher salt
concentration.
1964 Guha and Maheshwari produced the 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 and co-workers successfully achieved protoplast fusion.
1971 Takebe and co-workers regenerated the first plants from protoplasts
1972 Carlson produced the first interspecific hybrid of Nicotiana tobacum by
protoplast fusion.
1974 Reinhard introduced biotransformation in plant tissue cultures.
1977 Chilton and co-workers successfully integrated Ti plasmid DNA from
Agrobacterium tumefaciens in plants.
1978 Melchers and co-workers carried out somatic hybridization of tomato
and potato to develop pomato.
1981 Larkin and Scowcroft introduced the term somaclonal variation.
1983 Pelletier and co-workers conducted intergeneric cytoplasmic
hybridization in Radish and Grape.
1984 Horsh and co-workers developed transgenic tobacco by transformation
with Agrobacterium.
37
1985 Gheysen and co workers developed a very efficientgene transfer
system using Agrobacterium tumefaciens.
1986 Crossway and his coworkers developed a direct way of transferring
cloned genes into nucleus of tobacco mesophyll protoplasts by
microinjection of DNA.
1986 Kinsara and his coworkers produced somatic hybrids between
Lycopersicon esculentum and L. peruvianum.
1987 Terada and his coworkers regenerated plantlets from somatic hybrid
cells of Oryza sativa, and Echinochloa oryzicola.
1987 Klien and co-workers developed a biolistic gene transfer method for
plant transformation.
1987 Neuhaus and his coworkers achieved gene transfer by microinjecting
the DNA into the cells of microspore derived proembryos.
1988 Rhodes an his coworkers produced transgenic maize by
electroporation.
1988 Toriyama and his coworkers produced transgenic rice by
electroporation.
1989 Shimamoto and his coworkers produced fertile transgenic rice plants
from transformed protoplasts.
1990 Milanova and his coworkers succeeded in overcoming hybrid
incompatibility between Nicotiana africana and N. tabacum. They
produced cytoplasmic male sterile plants by embryo culture.
1990 Iida and his coworkers delivered genes into
cultured plant cells by DNA-coated gold particles accelerated by a
pneumatic particle gun.
1991 Kyozuka and his coworkers succeeded in getting tissue specific
expression of maize alcohol dehydrogenase l gene in transgenic rice
plants and their progenies.
1991 Sautter and his coworkers developed a novel method (called micro
targeting) for the acceleration of micro projectiles.
By the early 1980s techniques related to morphogenesis, somatic

embryogenesis were developed. In this period, culture of cereals, grasses,
legumes, and conifers that were previously considered to be recalcitrant
groups was successfully done. Interspecific and intergeneric hybrids were
produced using in vitro pollination. Plant tissue culture methods were
developed for overcoming sexual self-incompatibility and induction of haploid
plants. Embryo, ovary, anther and ovule cultures were successfully done for
large number of plant species. Protoplasts cultures have been developed for
more than more than 100 species of angiosperms.
Later in 1990s tissue culture techniques were developed for many types of
plants including cereals and grasses, legumes, vegetable crops such as
potato, root, tuber crops, oilseeds, temperate/ tropical fruits, plantation crops,
forest trees and ornamentals. Cryopreservation technology for storage of
germplasm was also developed. Plant tissue culture studies prove very useful
in understanding the concepts of morphogenesis, cytodifferentiation,
38 organogenesis and embryogenesis.
Genetic modification of plants by direct DNA transfer via vector-independent
and vector-dependent means has been achieved during this period. Vector-
independent methods such as electroporation, liposome fusion, microinjection
and high-velocity microprojectile bombardment (biolistics) were developed.
Advances in molecular biology proved useful in genetic engineering of plants
via insertion of foreign genes from diverse biological systems. The
development of shuttle vectors for harnessing the natural gene transfer
capability of Agrobacterium, use of these vectors for direct transformation of
regenerable explants and the development of selectable markers has been
focused in the recent years. The current emphasis and importance of plant
tissue culture is the improvement of plants. Over 100 species of plants have
been genetically engineered including dicotyledonous crops, few
monocotyledonous as well as some woody plants. Rice genome was
sequenced under International Rice Genome Sequencing Project in 2005. In
addition, technical improvements such as increased transformation efficiency,
commercial germplasm storage and low production costs for transgenic plants
have been done in the recent years.
Box 11.1 Contribution of Indian scientists in Plant tissue culture
In India, the plant tissue culture was initiated during 1950s at the University of Delhi.
Panchanan Maheshwari showed the production of haploid plants under in vitro
conditions. S.C. Maheshwari and Shipra Guha also made a remarkable contribution
to plant tissue culture by developing haploid plants from cultured anthers of Datura
innoxia that opened the new area of androgenesis. Techniques of in vitro culture of
floral and seed parts were developed during this period. Endosperm cultures and
plantlet regeneration via organogenesis were achieved later.
SAQ 1
Fill in the blanks with appropriate words.
a) The German Botanist ………………… first proposed the importance of

plant tissue and cell culture.
b) ………………….. cultured embryos of plants like Raphanus sativus.
c) …………………… demonstrated the possibility of raising large numbers

of plantlets from pollen.
d) ………………… first isolated protoplast by enzymatic degradation of the

cell wall.
e) ……………………. regenerated plants from protoplasts.
11.3 EQUIPMENTS REQUIRED FOR PLANT

TISSUE CULTURE
There are a few essential equipments that are required for the plant tissue
culture technique. A plant tissue culture laboratory needs to have an aseptic
chamber for culture and culture rooms or incubators. 39
Some of the basic equipments required for the tissue culture laboratory
include:
Laminar airflow cabinets: The most important aspect to be taken care of in

tissue culture is to maintain a contaminant-free environment within the lab.
A laminar airflow cabinet provides a place free from dust particle/micro
contaminants and helps in carrying out cell culture under an aseptic
environment. These cabinets help in maintaining an aseptic environment.
Autoclave machine: An autoclave machine is designed for sterilization. The

device will produce pressurized steam that helps in eradicating bacteria, fungi
and viruses. Autoclaves can be used for dry sterilization, i.e. sterilization of
scalpels and glass-wares such as Petri-dishes, pipettes etc. It is also used for
sterilization of the culture medium.
Water distillation apparatus: This is used for producing distilled water or

deionized water.
Culture rooms and/or Cabinets: The culture rooms/cabinets are equipped

with adjustable photoperiods including the intensity of light along with
temperature range so that controlled conditions can be maintained for the
cultivation of the cells.
Shakers: A rotary shaker or a reciprocal shaker is required for the

maintenance of suspension cultures.
A BOD incubator is required for maintaining a constant temperature to

facilitate the culture of microorganisms and their subsequent maintenance.
Fermentors or bioreactors are required to cultivate plant cells for the large-
scale production of chemicals or compounds. A pH meter is used for adjusting
the pH of the medium, a shaker to maintain cell suspension culture, a
weighing balance to weigh various components of the nutrient medium. A
freezer (used to store chemicals and stock solutions) is one of the basic
equipment found in a tissue culture laboratory. A spirit burner or gas micro
burner is needed for flame sterilization of instruments.
Incubation room or incubator provides the ideal environmental conditions

required for the growth and differentiation of cultured tissues. A typical
incubation chamber has light and temperature-controlled devices managed for
24 h period. AC or room heaters are required to maintain the desired
temperature range. Light needs to be adjusted in the terms of photoperiod
duration. Humidity in the range of 20-90% is required. Shelves are designed in
a way so that the culture vessels can be placed without any hindrance in the
light, temperature and humidity conditions.
Glassware is used to store cultures. Different kinds of culture vessels, large

test tubes (25×150 mm), wide mouth conical flasks are required in large
numbers for raising of cultures. In addition, graduated pipettes, measuring
cylinders, beakers, filters, funnel, and petri dishes are also required. Scissors,
scalpels and forceps are required for the preparation of explants from the
40 excised plant.
(a) (b)
(c) (d)
Fig. 11.1: The basic equipments used in plant tissue culture. a) photograph of an
autoclave; b) photograph of an incubator; c) photograph of a laminar
air flow; d) a view of culture room.
11.3.1 Basic Steps in Plant Tissue Culture

Plant tissue culture is an in-vitro culture of plant cells, tissues, or organs to
form a complete plant. In-vitro culturing of plants involves the following steps:
a) Selection and sterilization of explant:
A suitable explant is selected and is then excised from the donor plant. The
explant is then sterilized using disinfectants. An explant is surface sterilized
using mercuric chloride (0.11%), calcium hypochlorite (1-2 %) and alcohol
(70%). The tissue is immersed in sterilizing agent for 10 s to 15 min, and
thereafter washed with distilled water. 41
b) Preparation and sterilization of Culture Medium:
A suitable culture medium is prepared depending upon the type of explant to

be cultured. Culture medium prepared is transferred into sterilized vessels and
then sterilized in an autoclave.
c) Inoculation:
The sterilized explant is inoculated (transferred) on the culture medium under

aseptic conditions.
d) Incubation:
Cultures are then incubated in the culture room under appropriate controlled
conditions of light, temperature and humidity.
e) Subculturing:
Cultured cells are transferred to a fresh nutrient medium to get plantlets.
f) Transfer of Plantlets:
After the hardening process (i.e., acclimatization of plantlets to the

environment), the plantlets are transferred to greenhouse or pots.
11.4 COMPOSITION OF MEDIA

Various media have been tried for raising different cultures using different
explants. A suitable culture medium is required for culturing cells, tissues,
protoplasts, embryos, anthers, root tips, from the plant. The explant is a part of
the plant like root, stem, leaf, or meristematic tissue like cambium, floral parts
like anthers, stamens, etc. Growth and morphogenesis of the plant tissues in
in vitro conditions is governed by the composition of culture media. The
principal components of plant tissue culture media include:
• macro and micronutrients,
• carbon source,
• organic supplements such as vitamins, amino acids
• growth regulators and
• a gelling agent.
A complex nutritive medium composed of chemically defined compounds is

called a synthetic medium.
Although Knop’s solution was used. Later on, Murashige and Skoog (MS)
(1965) was developed as a new medium to induce organogenesis and
regeneration of plants. MS media consisted of macro-and micro-nutrients, a
carbon source, reduced N, B vitamins, and growth regulators. Another medium
was developed by Linsmaier and Skoog in 1965. The medium has a similar
component as Murashige and Skoog with some modifications of vitamins. It
was used for inducing organogenesis, callus culture, cell suspension, and
42 micropropagation. In 1968 O. L. Gamborg (Gamborg/B5) developed a
medium. This medium is a blend of nutrients like inorganic salts, vitamins, and
carbohydrates. The medium has a higher concentration of nitrate and
potassium and a lower concentration of ammonia. The media was used for
callus, cell suspension culture and protoplast culture.
Nitsch and Nitsch (NN) medium was developed by J. P. Nitsch in 1969 for the
establishment of in vitro anther culture of Nicotiana. It contains a high
concentration of thiamine, biotin, and folic acid. White’s medium was
developed by P. R. White in 1963. This culture media was developed for root
culture. It has a lower salt concentration and a higher concentration of MgSO4.
Another medium named N6 (Chu) was developed by Chu and co-workers in
1975 to culture anthers of rice plants. The medium promotes the initiation,
growth and differentiation of callus. The medium is a blend of inorganic salts,
vitamins, amino acids and carbohydrates. Media named EI was developed
specifically to culture soybean, red clover and other legumes. Haberlandt’s
media is used to produce whole plants (such as tobacco) from a single cell.
Various components of media help in the growth of explants. The

concentration of components of tissue culture media is expressed as mg/L or
ppm. According to the recommendations of the International Association for
Plant Physiology, the nutrients whose concentration should be greater than
0.5 g/L are macronutrients, while those nutrients whose concentration is less
than 0.5 g/L are called micronutrients. Hormones, vitamins and other organic
constituents are also generally added in concentrations less than 0.5 g/L.
Inorganic nutrients
These include macro and micronutrients. Macronutrients include elements

namely nitrogen, phosphorus, potassium, calcium, magnesium and sulphur
which are essential for plant cell and tissue growth. Micronutrients include iron,
manganese, zinc, boron, copper and molybdenum. The stock solutions of
macronutrients prepared are generally 10 times the strength of the working
medium, while stock solutions of micronutrients are made up at 100 times the
concentration of the working medium. Stock solutions are stored in a
refrigerator at 2- 4º C.
Carbon source
Sucrose is the most preferred external source of carbon used in the media.
Sucrose levels range from 2 to 6%.
Organic supplements
Thiamine (B1), nicotinic acid (B3) pyridoxine (B6), calcium pantothenate (B5)
and myoinositol are vitamins commonly used in tissue culture. Vitamins such
as thiamine, biotin, folic acid, riboflavin are also used. Vitamins are prepared
as either 100 or 1000 times concentrated stock solutions. Amino acids are
added to the media to stimulate the growth of cells, protoplast culture and
establish cell lines. Casein hydrolysate, L-glutamine, L-glycine, L-arginine and
L-cysteine are common sources of organic nitrogen used in the culture media.
Organic supplements such as protein hydrolysates, coconut milk, yeast
extract, malt extract, potato extract and tomato juice are also added to the
media. 43
In some media activated charcoal is also added. It also helps in reducing toxic
compounds produced during the culture and promotes the unhindered growth
of cells. Sometimes antibiotics such as Streptomycin and Kanamycin are
added in the culture media in very low concentrations to control systemic
infection.
Growth regulators
One or more hormones are added to culture media to promote organogenesis
and differentiation of tissues. Auxin and cytokinins play a role in cell division
and differentiation. Culture media is generally supplemented with auxins such
as indole acetic acid (IAA), naphthalene acetic acid (NAA), indole butyric acid
(IBA), 2, 4 diphenoxyacetic acid (2,4 D). GA3 is the most commonly used
gibberellin. It promotes the growth of cell cultures, enhances callus growth and
induces shoot elongation. The most commonly used cytokinins are 6 benzyl
amino purine (BAP), 6-benzyl adenine (BA), purine (zeatin). Balance of auxin
and kinetin in the medium influences morphogenesis. High levels of auxin
favour rooting while low levels help in the formation of the shoot and
intermediate levels to the proliferation of callus or wound parenchyma tissue.
Stock solutions of growth regulators are usually prepared at 100-1000 times
the final desired concentration and stored in a freezer (-20oC) for 2-3 months.
Gelling agent
A solidifying or gelling agent is added for preparing semisolid or solid media.
Agar, a compound obtained from seaweeds is generally used as a gelling
agent. Generally 0.5 to 1% of agar is added to the medium.
Iron source such as Iron EDTA is also added to the media. The stock solutions
of Fe are 100 times concentrated than the final working medium.
The pH of the media is also important as it affects the growth of plants and the
activity of plant growth regulators. It is adjusted to the value between 5.4 - 5.8.
Both the solid and liquid mediums can be used for culturing.
Full, half, or quarter strength media is prepared and this is based on the
explants, type of culture and many other conditions. The composition of some
commonly used media used in tissue culture has been given in Table 11.2.
Table 11.2: Composition of various Plant Tissue Culture Media.

Contents Type of media
MS White B5 Nitsch Heller NT
Inorganic
NH4NO3 1650 - - 720 - 825
KNO3 1900 80 2528 950 - 950
CaCl2.2H2O 440 - 150 - 75 220
CaCl2 - - - 166 - -
MgSO4.7H2O 370 750 247 185 250 1233
KH2PO4 170 - - 68 -- 680
(NH4)2SO4 - - 134 - - -
Ca(NO3)2. - 300 - - - -
44 4H2O
NaNO3 - - - - 600 -
Na2SO4 - 200 - - - -
NaH2PO2. H2O - 19 150 - 125 -
KCl - 65 - - 750 -
KI 0.83 0.75 0.75 - 0.01 0.83
H3BO3 6.2 5 10 3 1 6.2
MnSO4.4H2O 22.3 5 - 25 0.1 22.3
MnSO4.H2O - - 10 - - -
ZnSO4.7H2O 8.6 3 2 10 1 -
MoO3 - 0.001 - - - -
CuSO4.5H2O 0.03 0.01 0.03 0.25 0.03 0.25
CoCl2.6H2O 0.03 - 0.03 - - -
CoSO4.7H2O - - - - - 0.03
AlCl3 - - - - 0.03 -
NiCl2.6H2O - - - - 0.03 -
FeCl3.6H2O - - - - 1 -
Fe2(SO4)3 - 2.5 - - - -
FeSO4.7H2O 27.8 - - 27.8 - 27.8
Na2EDTA.2H2O 37.3 - - 37.3 - 37.3
Sequestrene - - 28 - - -
330 Fe
Organic
Inositol 100 - 100 100 - 100
Nicotinic acid 0.5 0.05 1 5 - -
Pyridoxine.HCl 0.5 0.01 1 0.5 - -
Thiamine HCl 0.1 0.01 10 0.5 - -
Glycine 2 3 - 2 - -
Folic acid - - - 0.5 - -
Biotin - - - 0.05 - -
Sucrose 3% 2% 2% 2% - -
D Mannitol - - - - - 12.70%
The constituents are given in the unit mg/L.
SAQ 2
Answer in one word
a) The part of the plant used in tissue culture.
b) Chemical used for the surface sterilization of the explants.
c) The primary source of carbon used in the media.
d) Exogenous balance of these hormones in the medium influences the

morphogenic fate of callus. 45
e) A relatively high level of this plant hormone favors rooting.
f) The medium containing a blend of nutrients like inorganic salts, vitamins,

and carbohydrates. The medium has a higher concentration of nitrate
and potassium and a lower concentration of ammonia.
g) Nutrients required in concentrations less than 0.5 g/L.
11.5 TOTIPOTENCY
In plant tissue culture technique, an explant is taken that is cultured on a
nutrient medium under the controlled conditions and develops into a whole
new plant. The potential of a plant cell to grow and develop into a whole new
multicellular plant is described as cellular totipotency. It depends upon the
inherent capacities of plant cells for differentiation. In other words, the capacity
of a cell to get differentiated into other cell types is called totipotency. The term
totipotency was coined in 1901 by Morgan. Totipotency of cells was first
hypothesized in the mid-19th century based on the regeneration capacity of
plants. In 1953, Muir was able to regenerate plants from isolated cells,
demonstrating the theory of cellular totipotency.
The potential of totipotency depends upon the differentiation potential of cells.

The cell undergoes dedifferentiation and re-differentiation (two inherent
processes). Differentiation (cyto-differentiation) is the phenomenon that
involves reverting of mature cells to dividing state. The ability of a
dedifferentiated cell to form a whole plant or plant organs is known as
redifferentiation. Differentiation of one organ directly from another organ is
known as trans-differentiation. To express totipotency, a differentiated cell first
undergoes dedifferentiation followed by re-differentiation.
Different plant parts and plants show different totipotent abilities. For e.g.
mostly somatic cells in the plant body are totipotent.
Various applications of cellular totipotency are:
• Crop improvement
• Micro-propagation of commercially important plants
• Production of artificial or synthetic seeds
• Conservation of germplasm (genetic resources)
• Production of haploids
• Production of somatic hybrids and cybrids
• Cultivation of plants whose seeds are very minute and difficult to

germinate
• High scale and efficient production of secondary metabolites
46 • Genotypic modifications
SAQ 3
Match the following:
Column A Column B
a) Totipotency 1. Murashige and Skoog medium
b) Unorganized mass of cells 2. Muir
c) Demonstrated the theory of 3. Morgan

cellular totipotency
d) Most extensively used medium 4. Callus

in tissue culture
e) Complex nutritive medium 5. Synthetic medium
11.6 ORGANOGENESIS
The development of organs like roots, shoots, and flowers directly from an
explant or from the callus is called organogenesis. In other words, it is the
development or regeneration of a complete organized structure i.e. whole
plant from the cultured cells/tissues. Organogenesis occurs due to stimulus
produced by the components of culture medium, substances present in the
explants and compounds produced during culturing.
The hypothesis of organogenesis was given by Torrey (1966). He suggested

that organogenesis starts with callus formation via the development of a group
of meristematic cells i.e. meristemoids. The meristemoids initiate the formation
of primordium which forms either shoot or embryoid. Shoot bud differentiation
was first demonstrated by White (1939). Organogenesis may occur either
through callus formation (indirect) or through the formation of adventitious
organs (like adventitious shoot) (direct) (Fig. 11.2). The direct mode of
organogenesis does not involve the formation of callus. Organogenesis has
been obtained from the culture of various explants such as cotyledons,
hypocotyl, stem leaf, shoot apex, root, young inflorescence, flower petals,
petiole and embryos. Hence the process of organogenesis involves two steps:
dedifferentiation and redifferentiation. Dedifferentiation results in the formation
of callus from the explant tissue whereas redifferentiation causes the
development of primordia from a group of callus cells.
The two main steps of Organogenesis are caulogenesis and rhizogenesis.

The initiation of root formation (adventitious roots) is termed rhizogenesis
while the initiation of shoot formation is called caulogenesis. Caulogenesis
starts with the induction of adventitious shoot buds. Caulogenesis is induced
by the optimal concentration of cytokinin present in the media. Rhizogenesis is
induced only after caulogenesis. 47
Fig. 11.2: Diagrammatic representation of various steps involved in

organogenesis.
Various culture media are used for organogenesis include MS (Murashige and
Skoog (1962), B5 (Gamborg (1969), white’s medium (White, 1963) and SH
(Schenk and Hildebrandt, 1972).
The process of organogenesis is regulated by various chemical and physical

factors such as physiological state and size of explants, medium and
environmental conditions such as temperature, quality and quantity of light.
High light intensity has been shown to inhibit shoot-bud formation. Blue light
promotes shoot-bud differentiation while red light stimulates rooting.
Temperature up to 33° C supports growth of callus but lower temperature (18º
C) stimulates differentiation of shoot-bud.
The concentration of growth regulator is critical for morphogenesis. Skoog and

Miller (1957) were the first to demonstrate the role of growth regulators in
morphogenesis. Skoog indicated that organogenesis is chemically controlled
i.e. chemical supplements in media regulate the process of organogenesis.
The formation of root and shoot is affected by the proportion of auxin and
cytokinins. A moderate to a high concentration of auxin such as 2, 4-D induces
callus formation by promoting the proliferation of cells. A relatively high
concentration of auxin favours cell proliferation and root differentiation while
high levels of cytokinin promote shoot bud differentiation.
Organogenesis proves very useful in understanding the mechanism of the

action of hormones (plant growth regulators), differentiation of cells and
tissues, development of plant organs, and other molecular mechanisms
48 involved in the plant.
SAQ 4
Differentiate between the following:
a) Dedifferentiation and Redifferentiation.
b) Rhizogenesis and Caulogenesis.
c) Direct and Indirect organogenesis.
11.7 EMBRYOGENESIS OR EMBRYOGENIC

DIFFERENTIATION
When embryos regenerate from somatic cells of plant organs, the process is
called somatic embryogenesis or embryogenic differentiation or
embryogenesis. Embryos formed from the somatic cells of the embryo sac (or
cells surrounding it) without gametic fusion under in-vitro conditions are called
somatic embryos or embryoids.
Induction of embryogenesis is achieved by two methods: direct and indirect

method. Direct embryogenesis involves the development of embryos directly
from the cells of explants or immature embryos. In indirect embryogenesis, an
intermediate step of callus growth is involved in the process. Explants are
cultured in a media containing growth regulators to produce embryogenic
callus which is later transferred to the proliferation medium.
Fig. 11.3: Diagrammatic representation of various steps involved in the formation of

plants from somatic embryos.
Somatic embryogenesis was first observed by Steward and his coworkers

(1958) in carrot (Daucus carota). Thereafter, somatic embryoids were obtained
from many plants namely Citrus, Coffea, Zea mays, etc. Embryoids can be
produced in specialized nutrient media. The process of formation of somatic 49
embryos has three main steps which include the induction of embryogenesis,
embryo development, and embryo maturation (Fig.11.3).
Embryogenesis is affected by physiological conditions, genotype of the plant

and type of explants. Auxin plays an essential role in the first step of
embryogenesis.
Importance
Somatic embryos (SEs) act as a valuable source for the large-scale
propagation of germplasm resources. The process of somatic embryogenesis
doesn’t involve the steps of fertilization (sexual fusion of gametes) and thus,
facilitates the rapid large-scale propagation of the plants. It also acts as a
powerful tool for cryo-storage of the embryo and valuable germplasm.
SAQ 5
Differentiate between direct and indirect embryogenesis.
11.8 PROTOPLAST CULTURE

A plasma membrane-bound vesicle formed as a result of removal of the cell
wall is called a protoplast. In vitro fusion of plant protoplasts derived either
from a somatic cell of the same plant or from two genetically different plants is
called somatic hybridization. The protoplasts are generally isolated from leaf
and mesophyll cells. The fused protoplast (parasexual hybrid protoplast) is
grown in vitro to produce a plant. The protoplast can be cultured and
regenerated into a whole plant. Takebe and his co-workers obtained tobacco
plants from mesophyll protoplasts.
J. Klercker (1892) first mechanically isolated protoplast from the plasmolyzed

cell of water warrior (Stratiotes aloides). E. Kiister (1927) isolated protoplasts
from fruits of several plants like Solatium nigrum, Lycopersicon esculetum, etc.
Kuster used a physiological method that involved hydrolysis of the cell wall for
isolating protoplasts. E. C. Cocking (I960) first reported the enzymatic method
for isolation of protoplast in a large number from root tip cells of Lycopersicon
esculentum. He used a concentrated solution of cellulase enzyme, prepared
from cultures of the fungus Myrothecium verrucaria to degrade the cell wall. I.
Takebe, Y. Otsuki and S. Aoki (1968) used cellulase and macerozyme
sequentially (in two steps) for the isolation of mesophyll protoplast of tobacco.
J. B. Power and E. C. Cocking (1968) demonstrated for the first time that a
mixture of two enzymes (cellulase + macerozyme) can be used simultaneously
(one-step method) for getting isolation of protoplasts. I. Takebe, G. Labib, G.
Melchers (1971) reported the plant regeneration from isolated protoplast in
Nicotiana tabacum.
The protoplasts are isolated from the plants utilizing chemical or enzymatic
procedure. The cell wall is removed by mechanical or enzymatic methods.
Isolated protoplast is placed in a hypertonic solution of a metabolic sugar such
as mannitol (13%) to prevent plasmolysis (Fig 11.4). The isolated protoplasts
are purified and then tested for their viability. The viable protoplasts are
cultured under in-vitro conditions using a suitable nutrient medium (either a
50 liquid medium or a semisolid agar medium).
Fusion of isolated protoplasts can be done mechanically by bringing the
isolated protoplasts in intimate physical contact mechanically under
microscope using micromanipulator and perfusion micropipette. The fusion of
protoplasts can also be induced by some chemical agents called fusogens.
Some commonly used fusogens are Poly Ethylene Glycol (PEG), high
concentration of Ca2+, Dextran, Poly Vinyl Alcohol, and synthetic
phospholipids. The polymer binds with the lipid membrane of cells and thus
induces fusion. The protoplasts carry negative charges and repel one another.
A fusogen reduces electronegativity and induces fusion. Fusion of protoplast
can also be induced by an electric current of low strength which is followed by
an electric impulse of high intensity (100 KV/m) applied for microseconds.
Protoplast fusion or somatic cell hybridization produces a hybrid cell that

contains two nuclei of two different parental protoplasts. The presence of two
different parental nuclei in the hybrid cell, i.e. heterodikaryotic condition can be
distinguished using the Carbol fuchsin staining technique or conventional
aceto-orcein or aceto-carmine staining. Non-toxic fluorochromes (fluorescein
isothiocyanate or FITC) or (rhodamine isothiocyanate or RITC) can also be
used for this. Heterokaryon exhibits apple-green fluorescence when FITC is
used and red fluorescence when rhodamine isothiocyanate is used.
Protoplast viability is influenced by the growing conditions such as light,

photoperiod, humidity, temperature, nutrition, and water, age of the plant and
leaf.
(a) (b)
Fig. 11.4: a) Various steps in somatic hybridization; b) Flow diagram depicting

process of formation of somatic hybrid. 51
Importance of Protoplast Culture
• Protoplast culture has immense potentialities for the improvement of
different plant species and varieties having high economic values. The
practical applications of protoplast culture include:
• Somatic hybrids can be developed between two sexually incompatible

species. Thus it proves the best method with the potential of overcoming
the sexual incompatibility barrier between plants.
• Cybrids (hybrids) which contain the nuclear and cytoplasmic genome of

one parent and only the cytoplasmic genome of the second parent are
produced by protoplast fusion. Cytoplasmic male sterile (CMS) lines may
be developed through protoplast fusion.
• Isolated plant protoplasts act as suitable material for genetic and

cytoplasmic modification experiments.
• Protoplasts are the ideal target for direct gene transfer. Direct gene
transfer can be done by using isolated protoplasts. Transfer of genes for
disease resistance, N2 fixation, rapid growth rate, protein quality, frost
hardiness and drought resistance has been successfully done through
protoplast culture.
• In case of vegetatively propagated plants, genetic variation can be

induced through the protoplast fusion of two species, varieties, or two
different genera.
• Production of plants with greater hybrid vigour has been obtained using
somatic hybrids. Success has been achieved in apomictic development
of somatic hybrids through protoplast fusion in cases of orange (Citrus
spp.) and mango (Mangifera indica).
• Regeneration of plants can be done using protoplast culture in several

forest tree taxa. For e.g. Eucalyptus, Populus alba, Picea, Abies, Larix
and Pinus taeda.
• Protoplast fusion technology is used for the fusion of plant cells and
yeast cells and is important for the characterization of yeast artificial
chromosomes (YAC), a potential vehicle for gene transfer.
• Protoplast culture has been used for the improvement of cereal crops
like wheat and maize. Successful production of tetraploids and triploids
of orange (Citrus spp.) has been achieved through protoplast fusion.
Intergeneric hybrids have been developed through protoplast culture in
Brassica.
SAQ 6
Fill in the blanks with appropriated words;
a) A membrane-bound vesicle which consists of a naked cell formed as a

result of removal of the cell wall is called …………………. .
b) ………………… is the first reported enzymatic method for isolation of

52 protoplast from root tip cells of Lycopersicon esculentum.
c) An essential step in the isolation of protoplast is the removal of the

…………………… from the cell.
d) Fusion induced by some chemical agents called fusogens is called

……………………. .
e) Polyethylene glycol (PEG) has been identified as a potent chemical used

for ……………………. .
11.9 SUMMARY
• In the plant tissue culture technique, the cell, organs are grown under in-
vitro aseptic conditions by culturing explants in a nutrient medium.
• Plant tissue culture has a great significance in crop improvement

programs. It helps in getting large-scale production of plants in short time
duration and improving the growth of plants of economic importance.
• The principal components of most tissue culture media include inorganic

nutrients (macro and micronutrients), carbon sources, organic
supplements, growth regulators and a gelling agent. Besides these
vitamins, amino acids are also supplemented in the media.
• The potential of a plant cell to grow and develop into a whole new
multicellular plant is called cellular totipotency. It depends upon the
inherent capacities of plant cells for differentiation.
• Organogenesis is defined as the development of organs like roots,

shoots, and flowers directly from an explant or the callus. The process of
organogenesis involves two steps: dedifferentiation and redifferentiation.
• The somatic cells of plant organs develop into embryos which are called
somatic embryos. The process is called somatic embryogenesis or
embryogenesis. Somatic embryos are obtained indirectly (with the
formation of callus) or directly from the explant.
• The protoplast can be cultured and regenerated into a whole plant.

The first demonstration of the totipotency of protoplasts was by
Takebe and co-workers.

1. Describe in brief the various components of the tissue culture medium.
2. What is the role of plant growth hormones in tissue culture medium?
3. What is totipotency? Enumerate its applications.
4. Organogenesis is chemically controlled. Justify the statement giving a

suitable explanation. 53
5. Enumerate the various steps of protoplast culture.
6. Discuss the importance of the technique of protoplast fusion in tissue

culture.
11.11 ANSWERS
Self Assessment Questions
1. a) Guttlieb Haberlandt
b) Hanning
c) Guha and Maheshwari
d) Cocking
e) Takebe and co-workers
2. a) explants
b) mercuric chloride
c) Sucrose
d) auxin and kinetin
e) auxin
f) Gamborg (B5) medium
g) micronutrients
3. a) Morgan
b) Callus
c) Muir
d) Murashige and Skoog medium
e) Synthetic medium
4. a) Refer to Section 11.5.
b) Refer to Section 11.5.
c) Refer to Section 11.5
5. Refer to Section 11.6
6. a) protoplast.
b) E. C. Cocking
c) cell wall
d) Chemofusion
54 e) fusogen
Terminal Questions
Acknowledgements
Fig. 11.1 : a) https://www.google.co.in/imgres?imgurl=https%3A%2F%2F
www.narang.com%2Fimages%2Fthumbnails%2Fautoclave
s-pressure-steam-
sterilizers.jpg&imgrefurl=https%3A%2F%2Fwww.narang.co
m%2Fautoclave-sterilizers%
b) https://www.google.co.in/imgres?imgurl=https%3A%2F%2F
5.imimg.com%2Fdata5%2FTA%2FGF%2FKO%2FSELLER
-3125581%2Flaboratory-incubators-500x500.jpg&imgrefurl
c) https://www.google.co.in/imgres?imgurl=http%3A%2F%2F
www.pureairsystemindia.com%2Finnovtouch%2Fuploads%
2F2021%2F07%2F2.png&imgrefurl=http%3A%2F%2Fwww
.pureairsystemindia.com%2Fproducts%2Flaminar-
airflow%2F&tbnid
d) https://www.google.co.in/imgres?imgurl=https%253A%252
F%252Fthumbs.dreamstime.com%252Fz%252Fplant-
tissue-culture-collection-shelves-tissue-culture-room-
science-laboratory-techniques-used-to-maintain-grow-
plant-
55
UNIT 12
PLANT TISSUE CULTURE (II
(II)
Structure
12.1 Introduction 12.6 Secondary Metabolite
Production
Objectives
12.7 Germplasm Conservation
12.2 Applications of Plant Tissue
Culture Methods of Germplasm
Conservation
12.3 Micropropagation
Applications of Germplasm
12.4 Haploid Production through
Storage
Androgenesis and
Gynogenesis 12.8 Summary
Applications of Haploids 12.9 Terminal Questions
12.5 Embryo Culture 12.10 Answers
Culture of Immature
Embryos/Embryo Rescue
Applications of Embryo
Culture
12.1 INTRODUCTION
In the previous units, we have discussed various techniques of tissue culture
and their uses. Tissue culture is a term used for methods that help in
maintaining and propagating plant cells and/or organs in vitro under aseptic
conditions. In tissue culture technique, the property of cellular totipotency
helps in getting differentiation of tissues, organs and the regeneration of the
entire plant. Clonal propagation of plants in a laboratory is done via
micropropagation. Explants obtained from a donor plant are cultured in sterile
conditions in tissue culture media. Explants develop into a complete plantlet
through somatic embryogenesis or organogenesis through callus formation.
This technique is used in genetic engineering to raise transgenic plants,
germplasm conservation, forestry, horticulture and many more.
In this unit, various applications of plant tissue culture such as conservation of

56 plant genetic resources, haploid production through androgenesis,
Unit 12 Plant Tissue Culture (II)
gynogenesis and production of secondary metabolites have been included. In
vitro methods have proved helpful in overcoming limitations found in
conventional methods. All the above-mentioned methods have helped in the
conservation of fast vanishing biodiversity.
Objectives
Objectives
enlist different ways in which plant genetic resources can be conserved;
have a complete understanding of pollen haploids and their applications

in agriculture and horticulture;
appreciate embryo culture and its applications;
differentiate between primary and secondary metabolites of plants;
describe the use of plant tissue culture for the production of secondary
metabolites;
describe the role of plant tissue culture in germplasm conservation; and
explain the technique of cryopreservation.
12.2 APPLICATIONS OF PLANT TISSUE

CULTURE
The tissue culture technique has been extensively used for vegetative
multiplication of plant species, getting virus-free plants, production of somatic
hybrids, genetic transformation and germplasm preservation. The process
involves growing/culturing plants cells in an agar medium to form the
undifferentiated mass of cells in the form of callus or as suspension culture
mass of free cells in a liquid medium (suspension culture). Some of the major
applications of plant cell and tissue culture are enumerated below:
1. Clonal propagation
The most widely used application of tissue culture technology is the vegetative
propagation of plants. Mass clonal propagation of plants can be achieved
using this technique. Clonal propagation refers to the multiplication of
genetically identical copies of individual plants (clones) through the process of
asexual reproduction. Clonal propagation involves the culture of apical shoots,
axillary buds and meristems on a suitable nutrient medium. Shoot meristems
can be regenerated in large numbers and thus many plantlets can be
produced (Fig. 12.1).
Clonal propagation is the rapid and large-scale production of plants from the
same genetic stock in a very short time. The technique of clonal propagation is
very useful commercially as several identical copies of plants can be obtained.
This technique is widely used in horticulture and silviculture. The plant material
can be provided in large quantities throughout the year irrespective of
seasonal variation. A large population of plant species with desirable traits can
be obtained using clonal propagation. This technique helps in providing 57
disease-resistant plants, multiplication of sexually derived sterile hybrids,
overcoming long seed dormancy of tree species. This technique also
facilitates the long-term storage of valuable germplasm. Clonal propagation
has been successfully done in plant species with dormant seeds, tree species,
orchids and many fruit plants. Apple, pears, strawberry, cardamom and many
ornamentals like orchids have been successfully propagated.
Fig. 12.1: Production of clones via clonal propagation.

2. Production of virus-free plants
Tissue culture has been used to produce virus-free plants for many crops and
Synthetic seeds or
ornamental plants at a commercial level. The meristems of in vitro raised
artificial seeds include
encapsulated somatic plants are generally free of infection, therefore meristem-tip culture is done to
embryos, shoot buds, obtain virus and pathogen-free plants. Virus-free plants of several
cell aggregates, or economically important plant species have been raised using the meristem
any other culture. For example: Potato virus X from potato, and mosaic virus from
meristematic tissue. cassava have been removed using the same plant tissue culture technique.
These seeds have
the ability to form a In vitro propagation of forest trees on a commercial scale has been done using
whole plant. The the technique of micropropagation. It has been successfully done for several
nutrients present economically important tree species like Acacia nilotica, Albizia lebbeck,
inside the Azadirachta indica, Butea monosperma, Dendrocalamus strictus, Shorea
encapsulation ensure robusta, Tectona grandis, Cedrus deodara, Cryptomeria japonica, Picea
and promote smithiana and Pinus sylvestris.
development of the
plants. 3. Production of synthetic seeds
Somatic embryos produced by tissue culture are encapsulated in a suitable
matrix (e.g. sodium alginate), along with substances like mycorrhizae,
insecticides, fungicides and herbicides to form “artificial seeds”. These seeds
can be utilized for rapid and mass propagation of desired plant species and
hybrid varieties. The synthetic seeds have several advantages such as :
i) easy to handle
ii) act as useful units of delivery
iii) can be easily stored for a long time without loss of viability
iv) directly sown in the soil like natural seeds without any acclimatization in
58 the green house.
4. Breaking Dormancy
The embryo (zygotic) culture technique helps in reducing the period of seed
dormancy. The breeding cycle can be shortened in many plants. Two
generations of flowering can be obtained in many plant species. For e.g. Rosa
sp., Malus sp, Ilex sp., Telia americana etc. The life cycle of Iris was reduced
from 2-3 years to less than one year using this technique.
5. Haploid Plants
Haploid plants can be obtained through anther or pollen culture

(androgenesis) or ovaries or ovule culture (gynogenesis). The anther culture
and haploid plant production has been done in many of the crop plants.
Haploids are of immense importance for the production of homozygous diploid
or polyploid lines without going into a series of backcrossing in a very short
period. This technique is of immense utility to a plant breeder as the time of
breeding gets reduced.
Haploids are sterile and possess a single set of chromosomes. They can be
converted into homozygous diploids by spontaneous or induced chromosome
doubling. The doubling of chromosomes helps in restoring the fertility of
plants. The double haploids become pure breeding new cultivars. The
production of haploids helps in overcoming the constraints of seed dormancy
and the non-viability of the embryo. Haploid plants show resistance to various
biotic and abiotic stresses and other desired traits.
6. Embryo rescue
In some plants, seeds take a long time to germinate and sometimes the seeds
do not germinate at all. Embryo culture can rescue this situation by embryo
rescue technique. The seeds are surface sterilized and split open in aseptic
condition and the tiny embryo is excised and planted in a nutrient medium and
then grows to a complete plant. Immature embryos can be recovered or
regenerated by tissue culture technique.
7. Production of Secondary Metabolites
Secondary metabolites are the cell constituents that are not essential for the
survival of plants but act as a good source of various compounds that are
useful at the commercial level. Plants act as the source of various
biochemicals which can be primary or secondary metabolites. These
compounds find their application in pharmacy, medicine and industry. These
metabolites possess antimicrobial, antibiotic, insecticidal, hormonal and
pharmacological properties. These include alkaloids, glycosides (steroids and
phenolics), terpenoids, latex, and tannins, etc. Secondary metabolites are the
most important chemicals produced by using cell culture. As the cells undergo
morphological differentiation and maturation during plant growth, some of the
cells specialize to produce secondary metabolites. Differentiated tissues
produce a high quantity of secondary metabolites under in vitro conditions.
The cultured cells produce biochemical compounds in high amounts under
appropriate culture conditions. The change in the physiological and
biochemical conditions enhances the production of these biochemicals in cells. 59
Enhanced productivity of secondary metabolites can be achieved via tissue
culture technique. Elicitors are the chemical compounds or molecules that
stimulate the production of metabolites. Elicitors can be of plant or microbial
origin. The elicitors of plant origin include polysaccharides derived from cell
walls for example – pectin, cellulose.
8. Genetic Variability/Somaclonal Variations

Plants regenerated from tissue and cell cultures show heritable variation for
both qualitative and quantitative traits. Such a variation is known as
somaclonal variation. This genetic variability is because of various ploidy
levels of cells and the genetic constitution of the explant. Different cultural
conditions can also induce such variations. Somaclonal variation may be
utilized in the improvement of crops since this reduces the time required for
the release of the new variety.
Such variations also show some useful characteristics such as resistance to a

particular disease, herbicide resistance, stress tolerance, etc. and some
agronomical traits like tiller number, panicle size, flowering time, plant height,
lodging resistance, yield, nutrient content, and different kinds of morphological
variations in leaf.
9. Somatic Hybrids
Somatic cell hybridization/ parasexual hybridization or protoplast fusion acts
as a good method for obtaining hybrids between species or genera with
desirable traits that cannot be produced via the conventional method of sexual
hybridization. Regeneration of plants by culturing protoplast in vitro has
opened a new avenue in various fields of plant breeding and plant
biotechnology. Products of fusion between two protoplasts (heterokaryon) are
cultured to regenerate a new somatic hybrid plant of the desired genotype.
Cybrid obtained by fusion of two protoplasts helps in the production of male
sterile line. Genes providing resistance to disease resistance such as Tobacco
mosaic virus, potato virus X, club rot disease can be successfully transferred
to species of interest.
10. Transgenic Plants

Transgenic plants are genetically modified (GM) plants in which a foreign gene
has been incorporated by the biotechnological procedure. Several transgenic
plants have been produced by incorporating genes for different traits such as
insect resistance, herbicide tolerance, delayed ripening, increased amino acid,
vitamin content and improved oil quality.
Various methods have been used to introduce foreign genes via plant tissue
culture. These include direct (electroporation, microinjection, or particle
bombardment) or Agrobacterium-mediated indirect processes. Important crops
can be improved by genetic engineering by isolating a specific gene and then
transferring it to selected crop plants.
11. Plant Germplasm Conservation
Germplasm refers to the sum of all the genes present in a crop and its related
species. The conservation of germplasm involves the preservation of the
60 genetic diversity of a particular plant or genetic stock for its use at any time in
the future. It is important to conserve the endangered plants or valuable
genetic traits present in the existing and primitive plants otherwise they will be
lost. The germplasm is preserved in the following two ways:
a) In-situ conservation - The germplasm is conserved in natural

conditions by establishing biosphere reserves such as national parks,
sanctuaries. This is used in the preservation of land plants in their
natural habitat along with several wild types.
b) Ex-situ conservation - This method is used for the preservation of

germplasm obtained from cultivated and wild plant materials. The
genetic material in the form of seeds or in vitro cultures is preserved and
stored as gene banks for long-term use.
12. Biomass production

The plant tissue culture has proved useful in improving the capacity of the
plant to produce more biomass by providing appropriate conditions such as
suitable culture media. Optimum culture conditions and nutrient medium
increase production of biomass by several folds. The enhancement in the
biomass production depends upon plant species, explants used and
composition of the medium. Various applications have been summarized in
Fig 12.2.
Genetic variability Secondary metabolites

Clonal propagation
Fast and large scale Preservation of germplasm

multiplication
Overcoming sterility Plant Tissue Disease free plants

Culture
Synthetic seeds
Somatic embryogenesis
Genetic transformation Somatic hybrids, cybrids
Fig. 12.2: An overview of various applications of plant tissue culture.
You will be made familiar with various techniques of germplasm conservation

in the subsequent sections of this unit.
SAQ 1
State whether the statements given below are True (T) or False (F):
a) The vegetative propagation of plants is a labour-intensive seasonal

process that is low in productivity.
b) The methods involved in the in vitro conservation of germplasm include

cryopreservation, low pressure and low oxygen storage, and cold
storage. 61
c) Virus-free plants in tissue culture are produced by nodal culture.
d) Somaclonal variation arises because of chromosome structural changes

like deletions, duplication, and gene mutations.
e) The major benefits of synthetic seeds include ease to store without

viability loss, rapid and mass propagation.
12.3 MICROPROPAGATION
Multiplication of genetically identical copies of a plant species by asexual
reproduction is called clonal propagation. In nature, clonal propagation
occurs by apomixis (seed development without meiosis and fertilization)
and/or vegetative propagation (regeneration of new plants from vegetative
parts). Tissue culture has become a popular method for the vegetative
propagation of plants. Clonal propagation under in vitro conditions is called
Micropropagation. A large number of the same types of plants can be
obtained in a relatively short time from a single individual. Micropropagation is
the only commercially viable method of clonal propagation for many
horticultural crops such as Orchids.
Micropropagation starts with the selection of plant tissues (explants or any part
of the plant such as leaf, apical meristem, bud and root) from a healthy,
vigorous parent plant. The whole process can be summarized into the
following stages as shown in Fig. 12.3.
62 Fig.12.3: A summary flow-chart of various steps involved in micropropagation.

Explants used in micropropagation
Different kinds of explants have been used in micropropagation. In orchids,
shoot tip (Anacamptis pyramidalis, Aranthera, Calanthe, Dendrobium,
Cymbidium, Odontioda), axillary bud (Aranda, Brassocattleya, Cattleya,
Laelia), inflorescence segment (Aranda, Ascofinetia, Neostylis, Vascostylis),
lateral bud (Cattleya, Rhynocostylis gigantean), leaf base (Cattleya), leaf tip
(Cattleya, Epidendrum), nodal segment (Dendrobium), flower stalk segment
(Dendrobium, Phalaenopsis) and root tips (Neottia, Vanilla) are being used in
micropropagation.
Various steps involved in the process of micropropagation have been listed

below:
Stage 0: Preparation of donor plant

Suitable plant tissue or explant is taken from the plant growing under natural
conditions and cultured under in vitro conditions.
Stage I: Initiation stage

In this stage, initiation and establishment of a culture in a suitable medium is
done. The explant is surface sterilized and transferred into a nutrient medium.
The surface sterilization removes contaminants with minimal damage to plant
cells. The most commonly used disinfectants are sodium hypochlorite, calcium
hypochlorite, ethanol and mercuric chloride. Thereafter, the cultures are
incubated in a growth chamber either under light or dark conditions.
Stage II: Multiplication stage

This stage mainly involves the multiplication of shoots or formation of embryo
from the explant. The cultures are maintained in growth chamber at of 20–
24° C and light intensity of 2000- to 4000-lux. During this stage, the number
of propagules is increased. This is done by repeated subcultures until the
desired number of plantlets are obtained.
Stage III: Rooting stage

This stage involves the transfer of shoots to a medium that help in the
formation of roots. The rooting is induced by addition of plant growth
regulators such as auxins in the media.
Stage IV: Acclimatization Stage

The plants raised under in vitro conditions are weaned and hardened.
Hardening is done by transferring the plant from conditions of high to low
humidity and from low light intensity to high light intensity. In this stage
plantlets are established in the soil. The plantlets of stage III from the
laboratory are transferred to the greenhouse. In case of some plant species,
shoots are directly planted in pots or suitable compost mix.
Advantages of micropropagation
i) Mass propagation of similar plants (clones) can be done through this
method. Instead of getting 10000 plants per year from an initial cutting in
vegetative propagation, we can get more than 1,000,000 plants per year
from one explant through micropropagation. 63
ii) Culture is initialized from small parts of plants and not much space is
required. From a small space of about 1 m2 in the culture room, about
20000 - 100000 plantlets can be produced per year.
iii) Disease and virus-free plantlets can be obtained.
iv) Micropropagation enables to increase production of plants that normally

propagate very slowly. Example bulbous crops and Narcissus.
v) Vegetative propagation of sterile hybrids can be done to get seed

production. For e.g. cabbage
Disadvantages of micropropagation
i) Expensive laboratory equipments are required.
ii) Plants are not autotrophic during culture conditions.
iii) The plants are poorly adapted to field conditions and need to be
acclimatized before transferring to field.
iv) There is a risk of genetic changes.
v) Mass propagation is not possible for all crops. For e.g. plants such as
cowpea is highly recalcitrant, and attempts to regenerate it using
in vitro-cultured explants have not been very successful.
vi) Adult woody plants cannot be easily regenerated via this technique.
vii) The induction of roots is not easily achieved in many species.
viii) Each explant has different in vitro growth rates and maturation, hence
uniform growth of plants cannot be obtained from tissue culture in the
case of many flowering plants.
SAQ 2
Fill in the blanks with appropriate words/terms:
a) ……………… is the crucial stage in tissue culture.
b) For producing virus-free plants, …………… is considered as the most

suitable explant.
c) Stage I in tissue culture is marked by………………...
d) Rooting occurs in ------stage of micropropagation.
e) The in vitro plants are weaned and need to be hardened in the

………………stage.
12.4 HAPLOID PRODUCTION THROUGH

ANDROGENESIS AND GYNOGENESIS
The higher plants are normally diploid, with two sets of chromosomes in their
64 somatic cells. Haploids (with one set of chromosomes) arise in nature by
parthenogenesis due to malfunction in the normal sexual process. Haploids
are sterile having only a single set of chromosomes and can be converted into
homozygous diploids by spontaneous or induced chromosome doubling.
Haploid plants are of great significance as they produce homozygous lines
(homozygous plants).
The production of haploids was first discovered in 1921 by Bergner in Datura

stramonium. The development of haploid embryos and plantlets from
microspores of Datura innoxia by the cultures of excised anthers was first
reported by Guha and Maheshwari. To date androgenic haploids of over 200
species including many major crop plants (cereals, mustards, potato, and
tomato) have been developed.
For the culture, anthers at the late uninucleate stage of microspore

development are excised from surface-sterilized buds and cultured on a
nutrient medium. A low temperature (4-5° C) treatment is given for initial 2-3
days to enhance the androgenic response. However, in some cases like
Brassica, a higher temperature (30-35° C) is required. The microspores
undergo repeated divisions to form multicellular structures. Such structures
directly develop into an embryo or form a callus from which plants are
regenerated via organogenesis or embryogenesis.
A summary of the standard protocol/procedure employed in raising the

androgenic haploids is outlined in Fig 12.4.
Fig 12.4: Diagrammatic representation of the production of plants by anther and

pollen culture.
In contrast to androgenesis which involves the development of an egg cell

containing a male nucleus into a haploid, a technique of gynogenesis is also
employed for in vitro production of haploids. It involves the culture of
unfertilized ovules whether egg (parthenogenesis) or any other haploid cells of
the embryo sac (apogamy) on media to produce an embryo bypassing the
process of fertilization. The gynobasic haploids were first developed from the 65
ovary cultures of Hordeum vulgare (Barley) by San Noem in1976. Haploids
through gynogenesis have been obtained in cereals like wheat, rice, and
maize and sugar beet, tobacco, sunflower.
The haploid plants obtained either by androgenesis or gynogenesis do not

possess a complete set of homologous chromosomes, they remain sterile and
do not form seeds. To obtain fertile plants, the haploids need to be made
diploid which results in the formation of fertile homozygous diploid plants.
12.4.1 Applications of Haploids

i) Using this technique, homozygous plants can be obtained in a relatively
short time as compared to the conventional breeding methods. The
doubling of chromosomes is achieved by colchicines treatment which
restores the fertility of plants. The double haploids show the potential to
become pure breeding new cultivars.
ii) Haploids help to detect recessive mutations which are normally not
expressed in heterozygous diploids.
iii) This technique also saves the plant breeders from the laborious and
lengthy procedure of inbreeding.
iv) In vitro androgenesis allows the breeders to detect gametoclonal

variation (variations occur due to recombination and segregation during
meiosis).
v) Anther culture has also contributed to the production of super males

(male populations with desirable features associated with male plants).
vi) Since the haploids show chromosomal instability, they can be used for
the introduction of alien chromosomes or genes in breeding programs.
Genetically transformed haploids have shown promising results by
exhibiting resistance to various biotic and abiotic stresses.
Box 12.1: Bulbosum technique
A technique popularly called “bulbosum technique” has been successfully used to

produce haploids in barley. The zygotic embryo culture of the tetraploid barley is
used to achieve this. A tetraploid Hordeum vulgare is first fertilized by a diploid H.
bulbosum. Zygotes of such hybrids are dissected out and the chromosomes of H.
bulbosum are eliminated, the remaining embryonal tissue is cultured on a nutritive
medium to produce dihaploid barley. Further crosses of this dihaploid produce the
progeny comprising haploids. Two varieties of barley, viz., ‘Gwylan” and “Mingo”
have been commercially produced by the bulbosum technique.
SAQ 3
a) Match the statements/terms given in Column A with those of Column B:
Column A Column B
i) Guha and Maheshwari 1. Androgenic haploids in

Datura
ii) Parthenogenesis 2. Embryo development from

66 an unfertilized egg
iii) Bulbosum technique 3. Chromosome elimination
following interspecific
hybridization
iv) Hordeum vulgare 4. gynobasic haploids
v) Colchicine 5. Double haploids
b) State whether the statements are True (T) or False (F).
i) Microspores and unfertilized eggs are known to form haploid

plants in culture.
ii) Haploids are important in genetic studies as they help to detect

recessive mutants.
iii) Haploids production by tissue culture is of academic interest only

as such plants are abnormal and cannot be integrated into
conventional breeding programs.
iv) Micropropagation allows the production of a large number of

propagules in a relatively short time throughout the year under
aseptic conditions.
12.5 EMBRYO CULTURE

Embryo culture involves the culture of an excised immature or mature embryo
from an ovule on a nutrient medium. The plant develops directly from the
embryo or indirectly through the formation of callus and then the subsequent
formation of shoots and roots. The technique has been very useful in:
• breaking seed dormancy,
• testing the vitality of seeds,
• production of rare species, haploid plants, and
• Shortening the breeding cycle of plants.
There are two types of embryo culture — mature embryo culture and immature
embryo culture (embryo rescue). Mature embryos are isolated from ripe seeds
and cultured in vitro when the embryos remain dormant for long periods or
there is low survival of embryos in vivo. This technique helps in overcoming
seed dormancy since the problem of dormancy due to inhibitors can be
overcome and sterile seeds can be converted into viable seeds. Embryo
culture is a relatively easy culture technique as embryos can be grown on a
simple inorganic medium supplemented with an energy source (usually
sucrose). This is because the mature embryos excised from the developing
seeds are autotrophic.
12.5.1 Culture of Immature Embryos/ Embryo Rescue

Embryo rescue involves the culture of immature embryos to rescue them from
becoming a part of unripe or hybrid seeds which fail to germinate (Fig. 12.5). 67
This approach is very useful to avoid embryo abortion and produce a viable
plant. Wild hybridization involving the crossing of two different species of
plants from the same genus or different genera often results in such a type of
failure. This is mainly because the normal development of zygote and seed is
hindered due to genetic barriers. Consequently, hybrid endosperm fails to
develop leading to the abortion of hybrid embryos. The endosperm may also
produce toxins that ultimately kill the embryo. Embryo abortions are due to
failure in endosperm development. Embryo abortion can be avoided by
isolating and culturing hybrid embryos. The most important application of
embryo rescue is the production of interspecific and inter-generic hybrids from
wild plant species.
Fig 12.5: Diagrammatic representation of embryo culture.
12.5.2 Applications of Embryo Culture

i) Incompatibility barriers in interspecific and inter-generic hybridization
programs leading to embryo abortion can be successfully overcome by
embryo rescue.
ii) Many distant hybrids have been obtained through embryo rescue
technique.
iii) Seed dormancy is caused by several factors endogenous inhibitors,

embryo immaturity, and physical factors like specific light and
temperature requirements, and dry storage requirements. Many plants
have a long natural period of seed dormancy. Embryo culture is
successfully applied to overcome seed dormancy and to produce viable
seedlings in such plant species.
iv) Some of the plants have long breeding cycles in their natural state. This
is mostly due to seed dormancy due to seed coat and/or endosperm.
The embryos can be excised and cultured in vitro to develop into plants
68 within a short period and can shorten the germination period.
v) Embryo culture has been successfully used to produce haploid (or
monoploid) plants e.g. the Bulbosum technique in barley.
vi) Certain plant species produce sterile seeds that do not germinate. Seed
sterility is mostly associated with incomplete embryo development which
leads to the death of the germinating embryo. Using embryo cultures, it
is possible to raise seedlings from sterile seeds of early-ripening fruits.
For e.g., apricot, plum and cherry.
vii) Embryos are ideally suited for in vitro clonal propagation. Since the
embryos are juvenile with high regenerative potential, it is possible to
induce organogenesis and somatic embryogenesis from embryonic
tissues.
SAQ 4
a) State whether the following statements are True (T) or False (F).
i) Ovary culture was first reported by San Noem.
ii) Bulbosum technique is used to overcome seed dormancy.
iii) Embryo culture is a relatively easy culture technique as the mature

embryos excised from the developing seeds are autotrophic.
iv) The most important application of embryo rescue is the production

of interspecific and inter-generic hybrids from wild plant species.
b) Give the technical term for the following statement:
i) In vitro production of plant from pollen grains.
ii) In vitro production of plants from unfertilized egg cell.
iii) In vitro propagation of plants.
iv) Variation among the plants raised from pollen grains.
12.6 SECONDARY METABOLITE PRODUCTION

In 1873, Sach found that metabolism gives rise to many by-products in plants.
These compounds called secondary products have economical importance.
These metabolites are synthesized by plants in low amounts (<1% of dry
weight). The production of such compounds varies with the development stage
and the physiological status of the plant.
Considering the medical importance of many of these secondary metabolites,

in-vitro studies have been focused on the production of commercially valuable
chemicals. Plants are the main source of about 25% of pharmaceutical
compounds. More than 11% of the basic and essential drugs listed by WHO
are exclusively derived from flowering plants. Tissue culture techniques have
proven very useful as they increase the production of these secondary
metabolites by many folds. 69
A commercial-scale production of secondary metabolites can be done via
tissue culture technique. The problems like variation in quality, pricing,
seasonal variation and can also be avoided/overcome by this technique. Plant
tissue cultures also provide a viable alternative to protect valuable biodiversity.
Compounds like Vincristine and Vinblastine, have been obtained from
Catharanthus roseus. In addition, the anti-cancerous drug paclitaxel (Taxol),
podophyllotoxin, camptothecin, morphine, diosgenin have also been
successfully obtained from cultured plants.
The advantages of plant cell cultures in metabolite production are manifold

that are listed below:
• shortening of maturity time of plants.
• The small amount of plant material is needed to get large-scale

production of phytochemicals.
• a short time period is required to produce large amount of compounds
• Preservation of valuable natural resources.
• Suspension cultures to obtain a higher yield of the desired products/s.
• Potential for the production of hitherto unknown novel compounds by

recovering new routes of biosynthesis from mutant and deviant cell lines
by incorporation of latest techniques of metabolic engineering,
metabolite mapping and use of recombinant DNA technology.
• Possibility of conversion of specific substrates to more valuable products

by single or multiple-step enzymes activity.
Some of the notable examples of early tissue culture-based secondary

metabolite production are:
• Increased levels of nicotine production in Nicotiana tabacum.
• Anthocyanin in Euphorbia milii.
• Anthraquinones in Morinda citrifolia, Crocus sativa, Asperula,

GaliumSherardia.
• Shikonins in Lithospermum erythrorhizon.
• Phenolic compounds in Acer pseudoplatinus.
• L-hydroxyquiphenylalanine (L-Dopa) in Stizolobium hassjoo.
• Ferruginol in Salvia miltorrhiza.
• Sanguinarine in Papaver somniferum.
• Ajmalcine and serpentine in Catharanthus roseus.
• Rosmarinic acid in Coleus blumei.
• Diosgenin in Dioscorea deltoids.
70 • Ginseng in Panax ginseng.

Tissue culture production is used to get large-scale production of secondary
metabolites and chemicals useful at the industrial level. Bioreactor systems
have been developed that are capable of producing various chemicals and
getting biological reactions under controlled conditions. Bioreactors provide a
biologically active environment and chemical process can be carried out using
microorganisms or biochemically active substances derived from such
organisms.
Some of the secondary metabolites produced by tissue culture are listed in

Table 12.1.
Table 12.1: List of some important secondary metabolites produced

commercially using tissue culture.
Plant name Secondary metabolite

Adhatoda vasica Vasine
Artemisia annua Artemisnin
Azadirachta indica Azadirachtin
Capsicum annum Capsiacin
Panax notoginseng Ginseng saponin
Podophyllum hexandrun Podophyllotoxin
Taxus chinensis Taxane, Palitaxel
Lithospermum erythrorhizon Shikonin
Coscinium fenustratum Berberin
Cassia senna Sennosides
Brucea javanica Cathin
Catharanthus roseus Ajmalicine, Vincristine
SAQ 5
Match the plant names given in Section A with secondary metabolites given in
Section B.
Section A Section B
a) Bruncea annum 1. Vincristine
b) Papaver somniferum 2. Rosmarinic acid
c) Lithospermum erythrorhizon 3. Shikonin
d) Coleus blumei 4. Cathine
e) Catharanthus roseus 5. Sangunarine
12.7 GERMPLASM CONSERVATION

Indiscriminate denudation of forests, clearing of agricultural lands for other
purposes, uncontrolled exploitation of wild plants for their economic benefit 71
has destroyed many natural habitats. As a result, there has been a continuous
depletion of biodiversity and erosion of the gene pool. The number of wild
species is decreasing and many of them are either endangered or vulnerable.
Some wild species have already gone extinct. Efforts have been made at the
international level to control and minimize the depletion of naturally occurring
genetic resources.
The practice of saving and selecting seeds or vegetative propagules for the
next sowing season has been a very ancient practice, which was also a form
of germplasm conservation and management. Populations in ancient China
and India exercised forest conservation practices as early as 700 B.C. Since
the genetic diversity once lost can never be regained, alternative methods of
conservation of the endangered genotypes, viz., tissue culture (in vitro cell and
organ culture) and germplasm conservation offer high promise to safeguard
the genetic wealth.
Germplasm broadly refers to the hereditary material (total content of genes)

transmitted from one generation to the next. Germplasm provides the raw
material to the breeder. Thus, the conservation of germplasm has significance
in all breeding programs. This further necessitates the need to conserve the
genetic diversity of a particular species for future use.
Two global bodies, namely the International Board of Plant Genetic Resources
(IBPGR) and subsequently International Plant Genetic Resources Institute
(IPGRI) have been established for germplasm conservation. The main
objective of these organizations is to provide the necessary support for the
collection, conservation, and utilization of plant genetic resources for the
benefit of present and future generations.
12.7.1 Methods of Germplasm Conservation

Two approaches are generally followed for germplasm conservation:
1. In situ conservation implies the conservation of plant biodiversity within

its natural habitat along with its ecosystem. This approach is useful for the
preservation of land plants in their natural habitat along with wild relatives.
This approach has received attention because the plant breeders cannot
simulate this system in the laboratory. The conservation of wild and cultivated
species is done by retaining them in the National Parks, Sanctuaries,
Biosphere Reserves.
This approach has advantages as it permits species-pathogen interactions

and their coevolution. It is also a dynamic conservation method as it allows the
conservation of lesser diversity in a single site. In addition, in situ conservation
permits the preservation of many recalcitrant species (recalcitrant seeds are
the seeds that will not survive during drying and freezing in ex situ
conservation).
Some major drawbacks of in situ conservation include:
a) The risk of losing germplasm due to environmental hazards,

natural/man-made disasters,
b) High cost of maintenance of many genotypes, and
72 c) Non-readily availability of germplasm for use.

2. Ex-situ conservation - Ex-situ conservation is the chief method for the
preservation of germplasm obtained from cultivated and wild plant materials
which are rare or endangered. It is mainly used for crop plants. The genetic
materials in the form of seeds or from in vitro cultures (plant cells, tissues, or
organs) can be preserved as gene banks for long-term storage under suitable
conditions. Adequate knowledge of the genetic structure of plant populations,
sampling techniques, regeneration methods, and maintenance of varietal gene
pools, especially in cross-pollinated species is essential for the successful
establishment of gene banks.
Methods of ex-situ germplasm conservation include:
a) Seed Banks
b) Botanical Gardens
c) Arboreta
d) In vitro methods
e) Cryopreservation
During the onset of the Green Revolution, high-yielding varieties of rice and
wheat were being grown everywhere as these varieties had a uniform genetic
constitution and their agronomic yields were high. Cultivation of locally
adapted varieties declined drastically causing a concern arose amongst
scientists that this may lead to loss of genetic diversity of these crops. This led
to the establishment of various agricultural institutions that aimed at the
conservation of germplasm. In 1974, International Board for Plant Genetic
Resources (IBPGR, now called Bioversity International) was established to
coordinate global efforts to maintain a system of collection and conservation of
plant biodiversity, throughout the world. Earlier, the focus for ex situ
conservation was only for food crops and their wild relatives but since the last
few years, importance has been given to rare and threatened wild species as
well.
One of the most common and convenient methods of germplasm conservation

is in the form of seeds. Seeds occupy less space, allow easy transportation
and are economical to store. Seeds of plants behave differently when stored.
Their response to low moisture tolerance, desiccation and low temperature
influences their longevity and viability. Based on their storage behaviour,
seeds can be classified as orthodox, recalcitrant, or intermediate.
Orthodox seeds have low moisture content (20% or less on a wet basis) and
can further be dried (5% or less moisture content) without losing their viability.
These seeds are desiccation-tolerant and can remain viable for several years.
Storage of these seeds at low temperature or cryopreservation enhances their
viability. Orthodox seeds include most grains and legumes, Cashew
(Anacardium occidentale), Citrus aurantifolia, Lantana camara, guava
(Psidium guajava), Capsicum annum and Hamelia patens.
Recalcitrant (unorthodox/desiccation sensitive) seeds are short-lived and very

sensitive to desiccation. These cannot be dried below a critical moisture level 73
and cannot tolerate freezing. They do not undergo maturation drying before
being shed like most other species. Since they have high moisture content,
they encourage microbial growth and rapid deterioration. They cannot be
freeze-stored like normal seeds as the ice crystals produced cause freeze
injury. Recalcitrant seed-producing plants are mostly propagated vegetatively.
Some notable examples are avocado, cacao, coconut, jackfruit, lychee,
mango, rubber, tea, and some horticultural trees.
Intermediate seeds are tolerant to desiccation but at the same time are
sensitive to low temperature. The seeds of coffee (Coffea arabica), papaya
(Carica papaya), Citrus, Florida royal palm (Roystonea regia), and oil palm
(Elaies guineensis) are examples of intermediate type.
There are certain drawbacks in conservation by seeds:
i) Recalcitrant and intermediate seeds lose their viability with time.
ii) Seeds are susceptible to pathogen or insect infection.
iii) Distinct clones cannot be maintained, and
iv) Seed conservation is applied only to those plants that are propagated by
seeds and those that are propagated vegetatively.
To circumvent the above-mentioned shortcomings, in vitro methods for

germplasm conservation are employed which are very useful for vegetatively
propagated plants, species with recalcitrant seeds and genetically engineered
materials. This approach has several advantages:
i) Small space is required for the maintenance of even large quantities of

material.
ii) The germplasm can be maintained in a pathogen-free environment.
iii) The conserved material can be protected against natural hazards.
iv) A Large number of plants can be obtained from the germplasm stock.
v) Since the germplasm is maintained under aseptic conditions, the

material can be easily transported across national and international
borders.
Despite having so many advantages, the ex-situ methods suffer from a serious
drawback. By this method, we are freezing the evolutionary development in
plants that could have taken place in nature.
Various materials can be stored by an in-vitro method. These include isolated

protoplasts, cells from suspension or callus cultures, propagules, seeds, or
meristem tips. Each material shows different responses to repeated cultures.
Cell or callus cultures which are in an undifferentiated phase are more
susceptible to genetic changes like additions and deletions during
subculturing. During culture, somaclonal or gametoclonal variations can lead
to genetic heterogeneity. To minimize the frequency of subculturing, it is
preferred to maintain the material as plantlets or store them as somatic
74 embryos.
Three basic approaches are followed for the in vitro conservation of
germplasm. These are: cold-Storage, cryopreservation, and storage under
low-pressure and low-oxygen conditions. The selection of these methods
depends upon two broad parameters i) slow-growth systems and ii) systems at
zero-metabolism rate.
1. Cold Storage: It involves conservation of germplasm at a low and non-

freezing temperature (1-9° C). This temperature treatment slows down the
growth of plant. This is a cost-effective method for germplasm storage and
has been employed successfully for storage of in vitro developed grapes (Vitis
vinifera), strawberry (Fragaria sp), rye grass (Lolium), Lotus (Nelumbo
nucifera), alfalfa (Medicago sativa), raspberry, blueberry (Rubus sp.), white
and red clover (Trifoliumsp) and apple (Malus domestica) plants. Since the
plant material is not completely frozen therefore there is no cryogenic injury.
The plants have been preserved for several years by this method.
2. Cryopreservation: The word cryo is derived from the Greek word krúos, =
icy cold, chill, frost. It implies the storage of germplasm in sub-zero
temperatures. It is a safe and cost-effective method for long-term conservation
of structurally intact living cells and tissues. The plant cell and tissue cultures
are brought to a zero metabolism or non-dividing state by reducing the
temperature in the presence of cryoprotectants.
Sub-zero temperatures can be achieved by subjecting the tissue to any four of

the following: i) solid carbon dioxide (-79° C), ii) low-temperature deep freezers
(-80° C), iii) vapour phase nitrogen (-150° C), and iv) liquid nitrogen (-196° C).
Liquid nitrogen is the most preferred cryopreservative as the cells remain in a
completely inactive state and thus can be conserved for long periods. The
cells do not divide and remain genetically stable. The formation of ice crystals
inside the cells should be prevented as they cause injury to the organelles and
the cell. In addition, cells may also be damaged by a high intracellular solute
concentration. Leaking out of certain solutes during freezing may also pose
problems. The viability of cells may also get altered by exposure to
cryoprotectants.
Successful cryopreservation of the material is based on the survival rate of cell

and tissue and their ability to re-grow or regenerate into complete plants or
form new colonies. The genetic integrity of recovered germplasm should be
assessed to determine whether it is ‘true-to-type’ following cryopreservation.
Cryobionomics is the study of genetic stability in the cryopreserved plant
materials, where the fidelity of recovered plants can be assessed at
phenotypic, histological, cytological, biochemical, and molecular levels.
The process of cryopreservation involves the following steps:
i) Selection of material.
ii) Pre-treatments (Cryoprotective treatments, dehydration, vitrification,

encapsulation).
iii) Freezing (rapid, slow or stepwise).
iv) Storage
v) Thawing 75
vi) Reculture
vii) Measurement of survival/viability
viii) Plant regeneration.
i) The ability of the explant to survive in cryopreservation is greatly

influenced by the morphological and physiological characters. Although
the meristematic cells and suspension cell cultures, in the late lag phase
(phase in which little to no cell division occurs) or log phase (phase that
show cell doubling) are most suitable, other tissues like meristems,
embryos, endosperms, ovules, seeds, cultured plant cells, protoplasts,
calluses can also be used.
ii) Damage to cells by freezing or thawing can be prevented by using

certain compounds called cryoprotectants. Ice crystal formation is
prevented or reduced as these chemicals lower the freezing point and
supercooling point of water. Dimethyl sulfoxide (DMSO), glycerol,
ethylene, propylene, sucrose, mannose, glucose, proline and acetamide
are some of the commonly used cryoprotectants. Generally, a mixture of
cryoprotectants is more effective than using one for more effective
cryopreservation without causing damage to cells/tissues.
iii) Since cells respond differently to low temperature, specific freezing

methods are employed for cryopreservation.
a) Slow-Freezing Method: This procedure is employed to cryopreserve

cells with suspension cultures. The tissue is slowly frozen at a slow
cooling rate of 0.5-4°C/min from 0°C to -100°C, and then transferred to
liquid nitrogen. The advantage of the slow-freezing method is that some
amount of water flows from the cells to the outside, which promotes
extracellular ice formation. Intracellular freezing is prevented and as a
result, the plant cells get partially dehydrated and survive better.
b) Rapid freezing method: This technique involves treating the plant

material with liquid nitrogen. During the process, the temperature
decreases at the rate of -300° to -1000°C/min. Since the freezing is
rapid, the intracellular ice crystals are not formed within the cells. Shoot
tips and somatic embryos respond best to the rapid freezing technique
as they are small and have low water content. Other tissues may not
survive rapid freezing, and therefore, would require stepwise freezing.
c) Stepwise freezing method: This method is a combination of slow and

rapid freezing procedures and combines the advantages of both. It is
carried out in a stepwise manner. The plant material is first cooled to an
intermediate temperature and maintained there for about 30 minutes.
This is followed by plunging the material into liquid nitrogen for rapid
cooling. During initial freezing, ice is formed only outside the cells while
the cell protoplasm does not freeze. This unfrozen protoplasm
subsequently loses water to the outside due to vapour pressure
deficit.The stepwise freezing method has been successfully used for
cryopreservation of suspension cultures, shoot apices and buds as these
76 tissues show a better survival rate than when subjected to rapid freezing.
d) Dry freezing method: It has been observed that the non-germinated dry
seeds can survive freezing at very low temperatures in contrast to water-
imbibing seeds which are susceptible to cryogenic injuries. Cells frozen
after dehydration in a vacuum are found to have a better survival rate.
The dry freezing technique requires variable dehydration optimum for
different species.
e) Storage: In germplasm conservation maintenance of the frozen cultures

at a specific temperature is important. The frozen cells/tissues are kept
for storage at temperatures in the range of -70 to -196° C. However, with
temperatures above -130° C, ice crystal growth may occur inside the
cells which reduces the viability of cells.
f) Thawing: Thawing is usually carried out by plunging ampules containing

the frozen samples into warm water bath (at 37-45°C) with vigorous
swirling. Rapid thawing occurs at the rate of 500- 750°C min-1. This
protects the cells from the damaging effects of ice crystal formation.
After thawing (ice completely melts), the ampoules are quickly
transferred to a water bath at 20-25°C. This transfer is necessary to
protect cells from getting damaged if left for long in a warm (37-45°C)
water bath.
g) Reculture: Thawed germplasm is washed several times to remove

cryoprotectants. The material is then re-cultured in fresh medium
following standard procedures. Many a time, directly culturing the
thawed material without washing yields better results as certain vital
substances get released from the cells during freezing.
h) Measurement of survival/viability: The viability of the cryopreserved

material is checked using stains like FDA (fluorescein diacetate), Evan’s
blue and TTC (2,3,5-triphenyl tetrazolium chloride). The entire
cryopreserved material may not always regenerate into complete plants.
The proportion of material that survives and divides to regenerate
complete plants can be quantified by the following formula:
Number of cells/organs growing

Survival (%) = × 100
Number of cells/organs thawed
i) Plant Regeneration: The cryopreserved cells/tissues are carefully

nursed and grown by adding certain growth-promoting substances.
Appropriate environmental conditions are maintained to ensure
successful plant regeneration. A list of selected plants whose various
forms have been successfully cryopreserved is given in Table 12.2.
Table 12.2: List of some successfully cryopreserved plants.
Plant species Plant material

Oryza sativa Cell suspension
Glycine max
Zea mays
Nicotiana tabacum
Capsicum annum
77
Oryza sativa Callus
Capsicum annum
Zea mays Protoplast
Nicotiana tabacum
Solanum tubersosum Meristems
Cicer arietinum
Zea mays Zygotic embryos
Hordeum vulgare
Manihot esculentum
Citrus sinensis Somatic embryos
Daucus carota
Coffea arabica
Nicotiana tabacum Pollen embryos
Citrus sp.
Atropa belladona
Low-Pressure and Low-Oxygen Storage: These techniques are developed

as an alternative to cryopreservation and cold storage. These techniques are
useful for both short-term and long-term storage. Low-Pressure storage
involves reducing the pressure of gases in contact with the plant material by
reducing the atmospheric pressure itself, Low-Oxygen Storage involves a
reduction in oxygen by combining it with an inert gas like nitrogen. Low-
Pressure Storage reduces the activity of pathogenic organisms and inhibits
spore germination in the plant culture systems. These techniques have also
been useful for extending the shelf life of fruits, cut flowers, potted plants, and
cuttings.
Synthetic Seed Technology: One of the modern approaches for short-term

germplasm conservation is the production of synseeds (synthetic
seeds/artificial seeds), which are produced by encapsulating somatic embryos
in a protective coating. You have studied this technique of short-term non-
cryogenic preservation in Section 12.2.
12.7.2 Applications of Germplasm Storage

Some of the important applications of cryopreservation germplasm storage
are:
1. Maintenance of stock cultures does not require sub-culturing. No need to

extend viability.
2. An ideal method for long-term conservation of cell cultures that produce

secondary metabolites (e.g., medicines, biologically active compounds).
3. Storage of disease (pathogen)-free plant materials and their propagation

as per requirement.
4. Long-term maintenance of recalcitrant seeds.
78 5. Conservation of somaclonal and gametoclonal variations in cultures.

6. Conservation of plant materials from endangered species.
7. Conservation of pollen for enhancing longevity.
8. Storage of rare germplasms developed through somatic hybridization

and other genetic manipulations.
9. Reliable method for the selection of cold-resistant mutant cell lines for
developing frost-resistant plants, and
10. Establishment of germplasm banks for the exchange of information at

the national and international levels.
One of the major limitations of germplasm storage is that the process requires
costly equipment and well-trained personnel.
SAQ 6
Answer in one word.
a) The hereditary material (total content of genes) transmitted from one

generation to the next.
b) The conservation of plant biodiversity within its natural habitat along with
its ecosystem.
c) Seeds that are short-lived and sensitive to desiccation.
d) The storage of germplasm in sub-zero temperatures.
e) Compounds that prevent damage to cells by freezing or thawing.
f) The technique in which the plant material is treated with liquid nitrogen.
12.8 SUMMARY
• Tissue culture is a term used to collectively refer to different methods
which are used to maintain and propagate plant cells and/or organs in
vitro under aseptic conditions.
• Tissue culture techniques have many important applications for various

industrial uses as well as for the protection of plant biodiversity.
• The property of totipotency helps to achieve differentiation of various

tissues and organs and the regeneration of the entire plant in tissue
culture.
• The technique of micropropagation allows clonal propagation of plants in

a laboratory and involves various steps starting from procurement of
explants from a donor plant and its culture in sterile conditions in tissue
culture media culminating in its development into a complete plantlet
through somatic embryogenesis or organogenesis. 79
• Micropropagation is now routinely used in genetic engineering to raise
transgenic plants and in other fields like germplasm conservation,
forestry, and horticulture. These techniques can also be used to raise
virus-free plants through callus culture of meristem tips.
• Another important application is to produce secondary metabolites which

have many industrial uses, mainly in the pharmaceutical industry. Plants
produce many of these secondary metabolites and manipulations during
culture can augment the production.
• Conservation of plant genetic resources is needed to protect and/or

store germplasm of crop species, a wild relative of crops as well as wild
species which are rare or endangered. Various in situ or ex-situ methods
are employed for germplasm conservation. In vitro conservation is an
important method for ex-situ type of conservation. Cryopreservation
involves keeping the material at very low temperatures, which
suppresses all the metabolic activity while retaining viability.

1. Enumerate various applications of plant tissue culture.
2. Write a short note on artificial seeds.
3. What is micropropagation? What are the advantages of

micropropagation over the conventional methods of clonal propagation
of plants?
4. Give a brief account of embryo culture.
5. How can cell suspension cultures be used for the production of

secondary metabolites?
6. Discuss in vitro germplasm conservation. Compare the advantages and

disadvantages of in-situ and ex-situ germplasm conservation.
7. Describe the steps involved in the cryopreservation of germplasm.
8. Define the following terms:
i) explants
ii) callus
iii) androgenesis
iv) embryoids
v) cryoprotectants
vi) somaclonal variation
vii) germplasm
80 viii) secondary metabolites

12.10 ANSWERS
Self-Assessment Questions
1. a) True; b) True; c) False; d) True; e) True
2. a) Multiplication
b) shoot tip
c) Initiation of culture
d) III
e) acclimatization.
3. a) i) gynobasic haploids
ii) Double haploids
iii) Androgenic haploids in Datura
iv) Embryo development from an unfertilized egg
v) Chromosome elimination following interspecific hybridization
b) i) True; ii) True; iii) False; iv) True
4. a) i) True; ii) False; iii) True; iv) False
b) i) androgenesis
ii) parthenogenesis/gynogenesis
iii) micropropagation
iv) gametoclonal variation
5. a) cathin
b) sanguinarine
c) shikonin
d) Rosmarinic acid;
e) vincristine
6. a) Germplasm
b) In situ conservation
c) Recalcitrant
d) Cryopreservation
e) Cryoprotectants
f) Rapid freezing method 81

Terminal Questions
4. Refer to section 12.5.
8. a) Any part of a plant that is used for culturing is known as explant.
b) An unorganized mass of cells capable of differentiating and

developing into a whole plantlet is known as a callus.
c) Production of haploid plants via a process of anther culture is

known as androgenesis.
d) Embryoids are embryo-like structures formed in vitro cultures and

have the potential to develop into full-fledged plants.
e) The compounds that prevent damage to cells by freezing or

thawing are called cryoprotectants.
f) Heritable variations for both qualitative and quantitative traits found

in cultured cells are known as somaclonal variations.
g) Germplasm- It is the hereditary material (total content of genes)

transmitted from one generation to the next.
h) The chemical compounds that are produced by the plant cell but
are not directly involved in primary metabolic processes of a plant.
82

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BBYET-143

ECONOMIC BOTANY AND

Block Preparation Team

Course Coordinator : Prof. Amrita Nigam and Dr. Ravi Rajwanshi

• Mr. Manoj Kumar for CRC Preparation.

Indira Gandhi National Open University, 2022

describe the concept of origin of biotechnology;

list out the applications of biotechnology;

appreciate the role of biotechnology in improvement of plants;

enlist the major techniques and methods in plant biotechnology;

describe the importance of plant tissue culture; and

enumerate the major applications of plant tissue culture.

10.5 Applications of 10.9 Summary

The advent of Recombinant DNA technology has made development of

describe the concept of biotechnology;

discuss the basics of plant biotechnology;

enlist various methods in plant biotechnology; and

enumerate various applications of plant biotechnology.

10.2 GENERAL ACCOUNT

Various stages in the development of Biotechnology

The development of biotechnology can be categorized as

• Ancient biotechnology (8000–4000 BC): Early history of biotechnology

• Classical biotechnology (2000 BC; 1800–1900 AD): This era of

• Modern biotechnology (1977): In this period, genetic information in

The production of cheese, yogurt and bread from micro-organisms, alcoholic

In 1919, Hungarian engineer Karl Ereky coined the term “Biotechnology".

In 1920, Chaim Weizman tried to produce useful chemical compounds

At the end of the eighteenth century and the commencement of nineteenth

By the end of the nineteenth century, many microorganisms were discovered,

Later in 1960s and 70s molecular, cellular technologies began to emerge. In

Table 10.1: A summary of historical events in Biotechnology.

Peiod Time/Year Event

1953–1976 The discovery of the structure of DNA revolutionized

Modern biotechnology, thus has developed as a science with enormous

10.3 TYPES OF BIOTECHNOLOGY

Biotechnology has been used in developing genetically modified plants to

Biotechnology also finds its applications in industrial sector. It includes use of

Biotechnology is used in treatment of waste and prevention of pollution. These

Recently a new system of classifying Biotechnology has been adopted. In this

Table 10.2: Categorization of various branches of Biotechnology on the

Color designation Description

Fig. 10.2: Categorization of branches of Biotechnology on the basis of colour.

10.4 VARIOUS TECHNIQUES IN

• Genetic Engineering Technology

The technique involves the manipulation of the DNA of an organism by

• Protein Engineering Technology

The technique involves improvement in existing proteins or creating novel

• Cell and Tissue Culture Technology

In this technique, cells/tissues are grown under laboratory conditions to

• Antisense or RNAi Technology

RNA interference (RNAi), is a mechanism that involves use of small RNAs

Fig. 10.3: An overview of RNAi technique.

Computational analysis of biological data, e.g., sequence analysis

• Protein separation and identification techniques

Techniques such as electrophoresis, microarrays and blotting techniques that

The genome approaches are used to determine the biological function of

Fig. 10.4: Genome approaches used to determine the biological function of

10.5 APPLICATIONS OF BIOTECHNOLOGY

Biotechnological techniques have been used in the diagnosis of