TABLE OF CONTENT

S.NO CONTENT Pg.NO

1. INTRODUCTION 2

2. DAY 1 & 2 4

3. DAY 3 &4 6

4. DAY 5&6 8

5. DAY 7 10
 INTRODUCTION

Tissue culture is the in vitro aseptic culture of cells, tissues, organs or whole plant under controlled
nutritional and environmental conditions often to produce the clones of plants. The resultant clones
are true-to type of the selected genotype. The controlled conditions provide the culture an
environment conducive for their growth and multiplication. These conditions include proper supply
of nutrients, pH medium, adequate temperature and proper gaseous and liquid environment.
Plant tissue culture technology is being widely used for large scale plant multiplication. Apart from
their use as a tool of research, plant tissue culture techniques have in recent years, become of major
industrial importance in the area of plant propagation, disease elimination, plant improvement and
production of secondary metabolites.Small pieces of tissue (named explants) can be used to
produce hundreds and thousands of plants in a continuous process. A single explant can be
multiplied into several thousand plants in relatively short time period and space under controlled
conditions, irrespective of the season and weather on a year round basis. Endangered, threatened
and rare species have successfully been grown and conserved by micropropagation because of high
coefficient of multiplication and small demands on number of initial plants and space.
In addition, plant tissue culture is considered to be the most efficient technology for crop
improvement by the production of somaclonal and gametoclonal variants. The micropropagation
technology has a vast potential to produce plants of superior quality, isolation of useful variants in
well-adapted high yielding genotypes with better disease resistance and stress tolerance capacities.
Certain type of callus cultures give rise to clones that have inheritable characteristics different from
those of parent plants due to the possibility of occurrence of somaclonal variability ,which leads to
the development of commercially important improved varieties. Commercial production of plants
through micropropagation techniques has several advantages over the traditional methods of
propagation through seed, cutting, grafting and air-layering etc. It is rapid propagation processes
that can lead to the production of plants virus free .Coryodalisyanhusuo, an important medicinal
plant was propagated by somatic embryogenesis from tuber-derived callus to produce disease free
tubers. Meristem tip culture of banana plants devoid from banana bunchy top virus (BBTV) and
brome mosaic virus (BMV) were produced. Higher yields have been obtained by culturing
pathogen free germplasmin vitro. Increase in yield up to 150% of virus-free potatoes was obtained
in controlled conditions. The main objective of writing this chapter is to describe the tissue culture
techniques, various developments, present and future trends and its application in various fields.

MISSION

The Tissue Culture technology provides a unique opportunity to rapidly multiply the elite types in vegetative
propagated plants. A whole plant can be regenerated from a small tissue in a suitable culture medium under
controlled environment. The plantlets so produced are called tissue culture raised plants. These plantlets are
a true copy of the mother plant and show characteristics identical to the mother plant.

VISION

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 The production of exact copies of plants that produce particularly good flowers, fruits, or have
other desirable traits.
 To quickly produce mature plants.
 The production of multiples of plants in the absence of seeds or necessary pollinators to
produce seeds.
 The regeneration of whole plants from plant cells that have been genetically modified.
 The production of plants in sterile containers that allows them to be moved with greatly reduced
chances of transmitting diseases, pests, and pathogens.
 The production of plants from seeds that otherwise have very low chances of germinating and
growing, i.e. orchids and Nepenthes.
 To clean particular plants of viral and other infections and to quickly multiply these plants as
'cleaned stock' for horticulture and agriculture.
 Reproduce recalcitrant plants required for land restoration

DAY 1 & 2

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BASICS OF PLANT TISSUE CULTURE

In plant cell culture, plant tissues and organs are grown in vitro on artificial media, under aseptic
and controlled environment. The technique depends mainly on the concept of totipotentiality of
plant cells which refers to the ability of a single cell to express the full genome by cell division.
Along with the totipotent potential of plant cell, the capacity of cells to alter their metabolism,
growth and development is also equally important and crucial to regenerate the entire plant. Plant
tissue culture medium contains all the nutrients required for the normal growth and development of
plants. It is mainly composed of macronutrients, micronutrients, vitamins, other organic
components, plant growth regulators, carbon source and some gelling agents in case of solid
medium. Murashige and Skoog medium (MS medium) is most extensively used for the vegetative
propagation of many plant species in vitro. The pH of the media is also important that affects both
the growth of plants and activity of plant growth regulators. It is adjusted to the value between 5.4 -
5.8. Both the solid and liquid medium can be used for culturing. The composition of the medium,
particularly the plant hormones and the nitrogen source has profound effects on the response of the
initial explant.
Plant growth regulators (PGR’s) play an essential role in determining the development pathway of
plant cells and tissues in culture medium. The auxins, cytokinins and gibberellins are most
commonly used plant growth regulators. The type and the concentration of hormones used depend
mainly on the species of the plant, the tissue or organ cultured and the objective of the experiment.
Auxins and cytokinins are most widely used plant growth regulators in plant tissue culture and their
amount determined the type of culture established or regenerated. The high concentration of auxins
generally favors root formation, whereas the high concentration of cytokinins promotes shoot
regeneration. A balance of both auxin and cytokinin leads to the development of mass of
undifferentiated cells known as callus.
Maximum root induction and proliferation was found in Stevia rebaudiana, when the medium is
supplemented with 0.5 mg/l NAA. Cytokinins generally promote cell division and induce shoot
formation and axillary shoot proliferation. High cytokinin to auxin ratio promotes shoot
proliferation while high auxin to cytokinins ratio results in root formation. Shoot initiation and
proliferation was found maximum, when the callus of black pepper was shifted to medium
supplemented with BA at the concentration of 0.5 mg/l. Gibberellins are used for enhanced growth
and to promote cell elongation. Maximum shoot length was observed in Phalaenopsis orchids when
cultured in medium containing 0.5 mg/l GA3 .

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DAY 3 & 4

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TISSUE CULTURE IN AGRICULTURE
As an emerging technology, the plant tissue culture has a great impact on both agriculture and
industry, through providing plants needed to meet the ever increasing world demand. It has made
significant contributions to the advancement of agricultural sciences in recent times and today they
constitute an indispensable tool in modern agriculture.
Biotechnology has been introduced into agricultural practice at a rate without precedent. Tissue
culture allows the production and propagation of genetically homogeneous, disease-free plant
material.
Cell and tissue in vitro culture is a useful tool for the induction of somaclonal variation. Genetic
variability induced by tissue culture could be used as a source of variability to obtain new stable
genotypes.
Interventions of biotechnological approaches for in vitro regeneration, mass micropropagation
techniques and gene transfer studies in tree species have been encouraging. In vitro cultures of
mature and/or immature zygotic embryos are applied to recover plants obtained from inter-generic
crosses that do not produce fertile seeds.
Genetic engineering can make possible a number of improved crop varieties with high yield
potential and resistance against pests. Genetic transformation technology relies on the technical
aspects of plant tissue culture and molecular biology for:

 Production of improved crop varieties

 Production of disease-free plants (virus)

 Genetic transformation

 Production of secondary metabolites

 Production of varieties tolerant to salinity, drought and heat stresses

GERMPLASM CONSERVATION

In vitro cell and organ culture offers an alternative source for the conservation of endangered
genotypes. Germplasm conservation worldwide is increasingly becoming an essential activity due
to the high rate of disappearance of plant species and the increased need for safeguarding the
floristic patrimony of the countries.
Tissue culture protocols can be used for preservation of vegetative tissues when the targets for
conservation are clones instead of seeds, to keep the genetic background of a crop and to avoid the
loss of the conserved patrimony due to natural disasters, whether biotic or abiotic stress. The plant
species which do not produce seeds (sterile plants) or which have ‘recalcitrant’ seeds that cannot be
stored for long period of time can successfully be preserved via in vitro techniques for the
maintenance of gene banks.
Cryopreservation plays a vital role in the long-term in vitro conservation of essential biological
material and genetic resources. It involves the storage of in vitro cells or tissues in liquid nitrogen
that results incryo-injury on the exposure of tissues tophysical andchemical stresses.

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Successful cryopreservation is often ascertained by cell and tissue survival and the ability to re-
grow or regenerate into complete plants or form new colonies. It is desirable to assess the genetic
integrity of recovered germplasm to determine whether it is ‘true-to-type’ following
cryopreservation.
The fidelity of recovered plants can be assessed at phenotypic, histological, cytological,
biochemical and molecular levels, although, there are advantages and limitations of the various
approaches used to assess genetic stability.
Cryobionomics is a new approach to study genetic stability in the cryopreserved plant materials.
The embryonic tissues can be cryopreserved for future use or for germplasm conservation.

DAY 5 & 6

EMBRYO CULTURE

Embryo culture is a type of plant tissue culture that is used to grow embryos from seeds and ovules
in a nutrient medium. In embryo culture, the plant develops directly from the embryo or indirectly
through the formation of callus and then subsequent formation of shoots and roots. The technique
has been developed to break seed dormancy, test the vitality of seeds, production of rare species
and haploid plants. It is an effective technique that is employed to shorten the breeding cycle of
plants by growing excised embryos and results in the reduction of long dormancy period of seeds.
Intra-varietal hybrids of an economically important energy plant “Jatropha” have been produced

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successfully with the specific objective of mass multiplication. Somatic embryogenesis and plant
regeneration has been carried out in embryo cultures of Jucara Palm for rapid cloning and
improvement of selected individuals. In addition, conservation of endangered species can also be
attained by practicing embryo culture technique. Recently a successful protocol has been developed
for the in vitro propagation of Khayagrandifoliola by excising embryos from mature seeds. The
plant has a high economic value for timber wood and for medicinal purposes as well. This
technique has an important application in forestry by offering a mean of propagation of elite
individuals where the selection and improvement of natural population is difficult.

GENETIC TRANSFORMATION
Genetic transformation is the most recent aspect of plant cell and tissue culture that provides the
mean of transfer of genes with desirable trait into host plants and recovery of transgenic plants [63].
The technique has a great potential of genetic improvement of various crop plants by integrating in
plant biotechnology and breeding programmes. It has a promising role for the introduction of
agronomically important traits such as increased yield, better quality and enhanced resistance to
pests and diseases.
Genetic transformation in plants can be achieved by either vector-mediated (indirect gene transfer)
or vectorless (direct gene transfer) method. Among vector dependant gene transfer
methods, Agrobacterium-mediated genetic transformation is most widely used for the expression of
foreign genes in plant cells. Successful introduction of agronomic traits in plants was achieved by
using root explants for the genetic transformation. Virus-based vectors offers an alternative way of
stable and rapid transient protein expression in plant cells thus providing an efficient mean of
recombinant protein production on large scale.
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Recently successful transgenic plants of Jatropha were obtained by direct DNA delivery to mature
seed-derived shoot apices via particle bombardment method. This technology has an important
impact on the reduction of toxic substances in seeds thus overcoming the obstacle of seed
utilization in various industrial sector. Regeneration of disease or viral resistant plants is now
achieved by employing genetic transformation technique. Researchers succeeded in developing
transgenic plants of potato resistant to potato virus Y (PVY) which is a major threat to potato crop
worldwide. In addition, marker free transgenic plants of Petunia hybrida were produced using
multi-auto-transformation (MAT) vector system. The plants exhibited high level of resistance
to Botrytis cinerea,causal agent of gray mold.

DAY 7

PROTOPLAST FUSION

Somatic hybridization is an important tool of plant breeding and crop improvement by the
production of interspecific and intergeneric hybrids.
The technique involves the fusion of protoplasts of two different genomes followed by the selection
of desired somatic hybrid cells and regeneration of hybrid plants.
Protoplast fusion provides an efficient mean of gene transfer with desired trait from one species to
another and has an increasing impact on crop improvement. Somatic hybrids were produced by
fusion of protoplasts from rice and ditch reed using electrofusion treatment for salt tolerance.
In vitro fusion of protoplast opens a way of developing unique hybrid plants by overcoming the
barriers of sexual incompatibility.
The technique has been applicable in horticultural industry to create new hybrids with increased
fruit yield and better resistance to diseases. Successful viable hybrid plants were obtained when
protoplasts from citrus were fused with other related citrinae species.
The potential of somatic hybridization in important crop plants is best illustrated by the production
of intergeneric hybrid plants among the members of Brassicaceae.
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To resolve the problem of loss of chromosomes and decreased regeneration capacity, successful
protocol has been established for the production of somatic hybrid plants by using two types of
wheat protoplast as recipient and protoplast of Haynaldiavillosa as a fusion donor. It is also
employed as an important gene source for wheat improvement.

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