ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

Plant Biotechnology (Tissue Culture)

Plant tissue culture: is a collection of techniques used to maintain or grow plant cells,
tissues or organs under sterile conditions on a nutrient culture medium of known
composition. Plant tissue culture is widely used to produce clones of a plant in a method
known as micropropagation.

Basic terms used in tissue culture:

Plant cell culture based on the unique property of the cell-totipotency.

Cell-totipotency is the ability of the plant cell to regenerate into a whole plant under
the laboratory conditions using artificial nutrient mediums.

Explant is an excised piece of differentiated tissue or organ is regarded as an explant.

The explant may be taken from any part of plant body e.g., leaf, stem, root.

Callus is the unorganized and undifferentiated mass of plant cells is referred to as callus.
Generally, when plant cells are cultured in a suitable medium, they divide to form callus.

De-differentiation is the phenomenon of mature cells reverting to meristematic state to

produce callus is dedifferentiation.

Re-differentiation is the ability of the callus cells to differentiate into a plant organ or
a whole plant is regarded as re-differentiation.

Nutrient media vary in type and quantity of materials used and the choice depends on
the type of plant to be grown. Some have a better growth in solid media than in liquid
media. The best known nutrient media are: White’s medium, MS medium, B5 medium.
The ideal pH of the culture medium is 5.8, and if the pH values of more than 7 or less
than 4.5, there is inhibition of growth.

History of plant tissue culture:

1|Page
 ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

Year By Home What

1838 Schwann & Schleiden Cellular theory
1902 Haberlandt Proposed concept of in vitro cell culture also
proposed Totipotency
1904 Hanning Nearly a mature zygotic embryo developed into a
plant in vitro
1922 Kolte & Robbins Successfully cultured root and stem tips
respectively
1924 Mayer Callus formation on carrot root explants by use of
lactic acid
1925 Laibach Developed of inter-specific embryo in vitro
1926 Went discovered first plant growth hormone –Indole
acetic acid
1934 Gautheret In vitro culture of cambial tissues of different trees
and shrubs failed
1934 Kogl Identification of the first plant hormone, IAA,
leading to cell enlargement
1939 Gautheret et al. Established endless proliferation of callus cultures
1941 Overbeek Coconut Milk used for growth and development of
very young Datura embryos
1942 Gautheret Observation of secondary metabolites in plant
callus culture
1943 Braun Identified tumor-inducing principle of crown gall
tumors
1946 Ball First whole plants of Lupines and Tropaeolum from
shoot tips
1948 Skoog Kinetin could induce organogenesis in tobacco
plant
1957 Skoog & Miller Gave concept of hormonal control (auxin:
cytokinin) of organ formation
1958 Maheshwari In vitro culture of excised ovules of Papaver
somniferum
1958 Maheshwari & Regeneration of somatic embryos from nucleus of
Rangaswamy Citrus ovules
1960 Cocking Was first to isolate protoplast by enzymatic
degradation of cell wall
1962 Murashige & Skoog Developed MS medium with higher salt
concentration

2|Page
 ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

1964 Guha & Maheshwari Produced first haploid plants from pollen grains of
Datura (Anther culture)
1970 Power et al. Successfully achieved protoplast fusion
1971 Takebe et al. Regenerated first plants from protoplasts
1981 Larkin & Scowcroft Introduced the term somaclonal variation
1996 Hansen Development of ‘agrolistic’ method of plant
transformation

Importance of tissue culture

 In a relatively short time and space a large number of plantlets can be produced
starting from the single explant.
 Taking an explant does not usually destroy the mother plant, so rare and
endangered plants can be cloned safely.
 It is easy to select desirable traits directly from the culture setup (in vitro) thereby
decreasing the amount of space required, for field trials.
 Once established, a plant tissue culture line can give a continuous supply of young
plants throughout the year.
 The time required is much shortened, no need to wait for the whole life cycle of
seed development. For species that have long generation time, low level of seed
production, or seeds that readily do not germinate, rapid propagation is possible.
 In vitro growing plants usually free, from the bacterial and fungal diseases. Virus
eradication and maintenance of plants in virus free state. This facilitates
movement of plant across international boundaries.
 Plant tissue banks can be frozen and then regenerated through tissue culture. It
preserves the pollen and cell collections from which plants may be propagated.

3|Page
 ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

Types of tissue culture

Callus culture: Callus culture may be defined as production and maintenance of an

unorganized mass of proliferative cell from isolated plant cell, tissue or organ by
growing them on artificial nutrient medium in glass vials under controlled aseptic
conditions.

Organ culture: That may allow differentiation and preservation of the architecture. The
organ culture refers to the in vitro culture and maintenance of an excised organ
primordial or whole or part of an organ in way and function.

Single cell culture: Single cell culture is a method of growing isolated single cell
aseptically on nutrient medium under controlled condition.

Suspension culture: Suspension culture is a type of culture in which single cell or small
aggregates of cell multiply while suspended in agitated liquid medium. Suspension
cultures are used in induction of somatic embryos and shoots, production of secondary
metabolites, in vitro mutagenesis, selection of mutants and genetic transformation
studies.

Embryo culture: Embryo culture may be defined as aseptic isolation of embryo (of
different developmental stages) from the bulk of maternal tissue of mature seed or
capsule and in vitro culture under aseptic and controlled physical condition in glass vials
containing nutrient semisolid or liquid medium to grow directly into plantlet.

Anther culture: Androgenesis is the in vitro development of haploid plants originating

from potent pollen grains through a series of cell division and differentiation.

Pollen culture: Pollen culture is the in vitro technique by which the pollen grains
(preferably at the microscope stages) are squeezed from the intact anther and then
cultured on nutrient medium where the microspores without producing male gametes.

4|Page
 ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

Somatic Embryogenesis: Somatic embryogenesis is the process of a single or group of

cells initiating the development pathway that leads to reproducible regeneration of non-
zygotic embryos capable of germinating to form complete plants.

Protoplast Culture: It is the culture of isolated protoplasts which are naked plant cells
surrounded by plasma membrane which is potentially capable of cell wall regeneration,
cell division, growth and plant regeneration on suitable medium under aseptic condition.

Shoot tip and Meristem culture: The tips of shoots (which contain the shoot apical
meristem) can be cultured in vitro producing clumps of shoots from either axillary or
adventitious buds. This method can, be used for clonal propagation.

Explant Culture: There are variety of forms of seed plants viz., trees, herbs, grasses,
which exhibit the basic morphological units i.e. root, stem and leaves. Parenchyma is
the most versatile of all types of tissues. They are capable of division and growth.

Plant in vitro culture techniques

The promise of plant in vitro technologies in three major areas, namely micro
propagation, somatic cell genetics and generation of transgenic plant.

Micropropagation – Propagation in tissue culture (micropropagation) is, used to

develop high-quality clonal plants. The main advantages are attributed to the potential
of rapid, large scale propagation of new genotypes, the use of small amount of original
germplasm.

Somatic cell genetics – Contribution of in vitro methods to plant breeding i.e. somatic
cell genetics is most significant, mostly in terms of haploid production and somatic
hybridization.

Transgenic plants – Expression of mammalian genes or other plant gene is becoming

routine for several plant species. One of the successful approaches has been engineered
for resistance against insects, virus and other pathogens as well as herbicide.

5|Page
 ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

Advantages

 In a relatively short time and space a large number of plantlets can be produced
starting from the single explants.
 In the living plant the behavior of each part of tissue is strongly influenced by
correlative controls imposed by the rest of the plant by isolating it in vitro, the
nature of some of these correlative controls can be determined.
 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.

Applications

 Micro propagation is widely used in forestry and in floriculture. Micro

propagation can also be used to conserve rare or endangered plant species.
 A plant breeder may use tissue culture to screen cells rather than plants for
advantageous characters, e.g. herbicide resistance/tolerance.
 Large-scale growth of plant cells in liquid culture inside bioreactors as a source
of secondary products, like recombinant proteins used as biopharmaceuticals.
 To cross distantly related species by protoplast fusion and regeneration of the
novel hybrid.

6|Page
 ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

 To cross-pollinate distantly related species and then tissue culture the resulting
embryo this would otherwise normally die (Embryo Rescue).
 For production of doubled monoploid plants from haploid cultures to achieve
homozygous lines more rapidly in breeding programs, usually by treatment with
colchicine which causes doubling of the chromosome number.

MICROPROPAGATION

Micropropagation is one of the most popular techniques of tissue culture. It is the

practice of rapidly multiplying stock plant material to produce a large number of
progeny plants, using modern plant tissue culture methods. Micro propagation is used
to multiply novel plants, such as those that have been genetically modified or breed
through conventional plant breeding methods. It is also used to provide a sufficient
number of plantlets for planting from a stock plant which does not produce seeds, or
does not respond well to vegetative reproduction.

General Technique of Micropropagation

The process of plant micropropagation aims to produce clones (true copies of a plant in
large numbers). The process is usually divided into the following stages:

Stage 0: Pre-propagation Stage

The pre-propagation stage requires proper maintenance of the mother plants in the
greenhouse under disease and insect free conditions with minimal dust. Clean enclosed
areas, glasshouses, plastic tunnels and net covered tunnels, provide high quality explant
source plants with minimal infection. Collection of explants for clonal propagation
should be done after appropriate pre-treatment of the mother plants with fungicides and
pesticides to minimize contamination in the in vitro cultures. This improves growth and
multiplication rates of in vitro cultures. The control of contamination begins with the
pretreatment of the donor plants.

7|Page
 ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

The choice of explant depends on the methods of shoot multiplication to be followed.

All plant organs viz. nodal segment, inter-nodal segments, shoot tip, root tip. For axillary
bud induction, callus culture, somatic embryogenesis explants nodal segments,
internodes and leaves are collected.

Stage 1: Initiation of Aseptic Culture

 In this stage sterilization of explants and establishment of explants were done.

The plant organ used to initiate a culture is called explant. The choice of
explant depends on the method of shoot multiplication to be followed.
 For micropropagation work the explant of choice is nodes
 For callus culture work the explant of choice is internodes and leaves.
 For somatic embryogenesis the explant is internodes and leaves.

Stage 2: Multiplication of Culture

This is the most important stage and the rate of multiplication determines the largely
success of micropropagation system this can be achieved by:

1. Enhanced axillary branching

2. Adventitious bud formation
3. Through callusing

1. Enhanced axillary branching: The axillary bud present in the axil of each leaf either
develops into a single shoot or form a cluster of shoots in the presence of cytokinins
(BAP 1.0mg/l) in the medium.

2. Adventitious Bud Formation: Buds arising from any part other than the leaf axils
or shoot apex are called adventitious buds. It is a standard horticulture practice.

3. Through Callusing: Plant cells are totipotent. In tissue culture, the mass of
differentiated cells commonly known as callus. This either gives rise to shoot bud or

8|Page
 ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

bipolar structure resembling embryo (somatic embryo). This method is used when aim
is to induce variability especially in self-pollinating species with narrow genetic base.

Stage 3: In Vitro Rooting of Shoots

In vitro grown shoots lack root system. For induction of roots they were transferred to
rooting medium. For rooting half strength MS medium supplemented with 1.0mg/l
auxin was used. Stage

4: Hardening and Acclimatization of Tissue Culture Plantlets

This is the final stage and requires careful handling of plants. The transplantation from
completely controlled conditions should be gradual. This process of gradually preparing
the plants to survive in the field conditions is called acclimatization. The plants produced
in tissue culture, although green in color; do not prepare sufficient food for their own
survival. Also inside the culture vessels humidity is very high and thus the natural
protective covering of cuticle is not fully developed. Therefore, immediately after
transfer plants were maintained under high humidity. Optimum conditions were
provided to plants in green house.

Advantages of Micropropagation

Micropropagation has a number of advantages over traditional plant propagation

techniques:

 The main advantage of micropropagation is the production of many plants that

are clones of each other.  Micropropagation can be used to produce disease-free
plants.
 Micropropagation produces rooted plantlets ready for growth, saving time for the
grower when seeds or cuttings are slow to establish or grow.

9|Page
 ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

 It can have an extraordinarily high frequency rate, producing thousands of

propagules while conventional techniques might only produce a fraction of this a
number.
 It is the only viable method of regenerating genetically modified cells or cells
after protoplast fusion.
 It is useful in multiplying plants which produce seeds in uneconomical amounts,
or when plants are sterile and do not produce viable seeds or when seed can't be
stored.
 Micropropagation often produces more robust plants, leading to accelerated
growth compared to similar plants produced by conventional methods - like seeds
or cuttings.
 Some plants with very small seeds, including most orchids, are most reliably
grown from seed in sterile culture. A greater number of plants can be produced
per square meter and the propagules can be stored longer and in a smaller area.

Disadvantages of Micropropagation

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

 It is very expensive, and can have a labor cost of more than 70%.
 A monoculture is produced after micropropagation, leading to a lack of overall
disease resilience, as all progeny plants may be vulnerable to the same infections.
 An infected plant sample can produce infected progeny. This is uncommon if the
stock plants are carefully screened and vetted to prevent culturing plants infected
with virus or fungus.

10 | P a g e
 ADVANCE CELL BIOLOGY BY: HIWA HUSSEIN HASAN

FIGURE 1. Prunus africana in vitro propagation through axillary shooting from nodal segments in
optimum growth conditions. (A) Seeds of P. africana. (B) Maternal P. africana plant from
germinated seeds. (C) Axillary shoots formed on the P. africana nodal segment cultured in Woody
Plant Medium (WPM) with vitamins supplemented with 15 g L−1 sucrose and 1.0 mg L−1 6-
Benzylaminopurine (BAP). (D,E) Rooting of excised axillary shoots in WPM with vitamins
supplemented with 15 g L−1 sucrose, and 1.5 mg/L−1 indole-3-acetic acid (IAA). (F) In vitro rooted P.
africana plantlets. (G) Prunus africana plantlet planted in horticulture soil mixed with perlite in the
ratio of 2:1. (H) Acclimatized regenerated P. africana plant with well-developed root and shoot
systems in horticulture soil mixed with perlite in the ratio of 2:1. (I) Regenerated P. africana in
greenhouse.

11 | P a g e