Plant Biotechnology (Tissue Culture)

Report

By
PhD. Student: Hiwa Hussein Hasan

Supervised by
Asst. Prof. Dr. Treefa Farouq Ismail

2023-2024
Contents
Tissue Culture ......................................................................... 3

1 Basic terms used in plant tissue culture ......................... 4

2 History of plant tissue culture ......................................... 4

3 Types of plant tissue culture ........................................... 5

4 Plant in vitro culture techniques ..................................... 7

5 Environmental condition of plant tissue culture ......... 13

References ............................................................................. 15

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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. Different techniques in plant tissue culture may
offer certain advantages over traditional methods of propagation, including:

 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.

Plant tissue culture relies on the fact that many plant cells have the ability to
regenerate a whole plant (totipotency). Single cells, plant cells without cell walls
(protoplasts), pieces of leaves, stems or roots can often be used to generate a new
plant on culture media given the required nutrients and plant hormones.

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1 Basic terms used in plant tissue culture

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.

2 History of plant tissue culture

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

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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
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

3 Types of plant tissue culture

Plant tissue culture encompasses various techniques and methods, each serving
specific purposes in plant propagation, research, and biotechnology. Here are some
common types of plant tissue culture:

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1. Micropropagation:
 Shoot Culture: Involves the culture of shoot tips or axillary buds to produce
multiple shoots, which can then be rooted and grown into complete plants.
 Somatic Embryogenesis: Formation of embryos from somatic (non-
reproductive) cells, allowing the production of plants without the need for
seeds.
 Meristem Culture: Involves the culture of the apical meristem, the growing
tip of a plant, to eliminate viruses and produce virus-free plants.
2. Callus Culture:
 Callus Induction: Culturing undifferentiated cells from explants, often
leading to the formation of a mass of cells known as callus.
 Cell Suspension Culture: Suspension of single cells or small cell aggregates
in liquid medium, which allows for the production of biomass and extraction
of secondary metabolites.
3. Embryo Culture:
 Embryo Rescue: Involves the culture of immature or rescued embryos to
obtain viable plants, especially in cases where seeds may not develop or
mature.
4. Anther and Pollen Culture:
 Anther Culture: Culturing anthers (male reproductive structures) to induce
the formation of haploid plants.
 Pollen Culture: Culturing pollen grains to produce haploid plants, useful in
plant breeding and genetic studies.
5. Protoplast Culture:
 Protoplast Isolation: Removal of the plant cell wall to create protoplasts
(individual plant cells).

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  Protoplast Fusion: Fusing protoplasts from different plant species or varieties
to create hybrid plants with combined traits.
6. Hairy Root Culture:
 Agrobacterium rhizogenes Transformation: Inducing the formation of
transformed roots by infecting plant tissues with the bacterium Agrobacterium
rhizogenes, leading to the production of "hairy roots" that can produce
secondary metabolites.
7. Organ Culture:
 Organogenesis: Inducing the development of complete plant organs (such as
roots, shoots, or leaves) from explants.
 Apical Dominance Removal: Removing the apical meristem to encourage
lateral bud development and branching.
8. Secondary Metabolite Production:
 Plant Cell Culture for Metabolite Production: Using plant cells to produce
specific secondary metabolites of interest, such as alkaloids, flavonoids, and
phytochemicals, for pharmaceutical or industrial purposes.

These various types of plant tissue culture techniques are employed based on the
specific goals of researchers, plant breeders, and biotechnologists. Each method has
its advantages and applications, contributing to advancements in plant science,
agriculture, and biotechnology

4 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.

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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.

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

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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.

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

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 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
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.

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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.

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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.
 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:

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  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.

5 Environmental condition of plant tissue culture

The success of plant tissue culture depends on maintaining specific environmental

conditions that support the growth and development of plant cells and tissues. Here
are some critical environmental factors for plant tissue culture:

1. Sterile Environment:
 Sterility is crucial to prevent contamination by bacteria, fungi, and other
microorganisms. Tissue culture work is typically conducted in laminar flow
hoods or cleanrooms with controlled airflow and high-efficiency particulate air
(HEPA) filters.
2. Temperature:
 The temperature is a vital factor influencing plant tissue culture. It is usually
maintained between 20°C and 25°C for most plant species. However, specific
temperature requirements may vary depending on the plant species and the
stage of tissue culture.
3. Light:
 Light is essential for photosynthesis, but the intensity and duration of light
exposure depend on the plant species and the stage of tissue culture. Some
cultures may require continuous light, while others may benefit from a
photoperiod resembling day and night.
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4. Humidity:
 Maintaining high humidity levels is crucial to prevent desiccation of plant
tissues. This is particularly important during the early stages of culture when
explants are establishing themselves.
5. Ventilation:
 Proper ventilation ensures the exchange of gases such as carbon dioxide and
oxygen. Adequate airflow helps regulate temperature and prevents the
accumulation of ethylene, which can be detrimental to tissue culture.
6. pH Level:
 The pH of the culture medium is critical for nutrient availability and metabolic
processes. The optimal pH varies depending on the type of plant being cultured,
but it generally falls in the range of 5.5 to 6.5.
7. Nutrient Composition:
 The culture medium must contain essential nutrients, including macro- and
micronutrients, vitamins, and organic supplements. These components provide
the necessary elements for the growth and development of plant tissues.
8. Relative Humidity:
 Besides maintaining high humidity within the culture vessel, the overall
relative humidity in the laboratory or growth chamber should be controlled.
This helps prevent water loss from the culture medium and plant tissues.
9. Cultural Vessel and Sealing:
 The choice of culture vessel and its sealing are important. Containers should be
airtight to prevent contamination and evaporation. The type of vessel (e.g., jars,
test tubes, or culture flasks) may vary based on the specific needs of the culture.
10.Periodic Subculturing:
 Regular subculturing or transfer of cultures to fresh media is essential to
provide a continuous supply of nutrients and remove metabolic by-products.

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It's important to note that the optimal conditions can vary among different plant
species and even among different stages of tissue culture (initiation, multiplication,
rooting, etc.). Therefore, advancements in technology and equipment, such as
controlled environment chambers and automated systems, have improved the
precision and reproducibility of plant tissue culture.

References
1. Galun, Esra (2007). Plant Patterning: Structural and Molecular Genetic Aspects.
World Scientific Publishing Company. p. 333. ISBN 9789812704085
2. Leibfried, A., To, J. P., Busch, W., Stehling, S., Kehle, A., Demar, M., ... &
Lohmann, J. U. (2005). WUSCHEL controls meristem function by direct
regulation of cytokinin-inducible response regulators. Nature, 438(7071), 1172-
1175. doi:10.1038/nature04270
3. Kauth, P. J., Vendrame, W. A., & Kane, M. E. (2006). In vitro seed culture and
seedling development of Calopogon tuberosus. Plant Cell, Tissue and Organ
Culture, 85(1), 91-102. DOI 10.1007/s11240-005-9055-1
4. Bhojwani, S.S., & Dantu, P.K. (2013). Plant Tissue Culture: An Introductory Text.
Springer India
5. Kyte, Kleyn, et al (2013) Plants from test tubes: An introduction to
micropropagation. Timber press, Inc.
6. Sharma, V., & Alam, A. (2015). Plant Tissue Culture. I.K. International Publishing
House Pvt. Ltd.

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