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About the Editors
Science for Community Service and Environmental
Development for three consecutive years, Vice Dean of
Development and Quality at the Community College
for one year and Adviser to the Saudi Ministry of
Environment, Water and Agriculture and the National
Center for the Development of Vegetation Cover and
Combating Desertication for the second year in a row.
Muhammad Riaz is an Assistant Professor of
Pharmacy at Shaheed Benazir Bhutto University,
Sheringal, Pakistan. He has authored over 40 peerreviewed publications in renowned journals at the
national and international level and three books:
Anthocyanins and Human Health: Biomolecular and
Therapeutic Aspects (2016) and Carotenoids: Structure
and Function in the Human Body (2021) for Springer
and The covid-19 pandemic: A Multidisciplinary
Review of Diagnosis, Prevention, and Treatment
(2022) for CRC/Apple Academic. He received his PhD
in Pharmacognosy from the University of Karachi,
Pakistan (2012), followed by a postdoctoral fellowship
at Prof. Dou Deqiang Lab, Liaoning University of
Traditional Chinese Medicine, China (2017). During
his PhD, he worked at Prof. Michael Heinrich Labs,
UCL School of Pharmacy, London, (2010) under
International Research Support Initiative Program of
the Higher Education Commission of Pakistan.
Umar Farooq Gohar has a bachelor’s degree in
Pharmacy (B Pharmacy) from the University of the
Punjab and Master of Philosophy (MPhil) and Doctor
of Philosophy (PhD) in Biotechnology from the
Institute of Industrial Biotechnology, Government
College University, Lahore. Dr. Gohar did some of his
PhD research work at the School of Chemical and
Biomolecular Engineering, University of Sydney,
Australia. He has served Pharmacy Department at the
University of Lahore as Lecturer; later he served
Riphah International University as Assistant Professor.
Currently he is working as Assistant Professor at the
Institute of Industrial Biotechnology, Government
College University Lahore. Dr. Gohar is currently
supervising ve PhD scholars. He has also supervised
more than 40 MPhil projects in the eld of pharmacy
About the Editors
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xiii
and biotechnology. He has completed two projects
funded by ORIC Government College University and
Higher Education commission of Pakistan. He has
published more than 45 publications in various national
and international journals. He is reviewer of many
peer-reviewed journals. His area of interest is production and application of bioactive compound from
microbial and plant sources. He is also guest editor of
two special issues of journal Molecules. He also
remained associated with a biannual journal Biologia
Pakistan. He got Mevlana exchange program funding
from Uşak University, Turkey.
Chapter 1
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Tissue Culture ofMedicinal Plants
IshaFatima, MuhammadAkram, HamidMukhtar, UmarFarooqGohar,
ZahoorAhmadSajid, andUzmaHameed
1.1 Introduction
Plants are a major source of medicinally important metabolites. With the increasing
demand for medicines, the need to produce naturally occurring pharmaceutically
important metabolites of plants has become essential. Tissue culture or micropropagation proved to be the most appropriate method for the healthy cultivation of
medicinal plants. It is gaining popularity due to season-independent growth with
less time and space. Tissue culture techniques are employed to obtain plants in a
short time and a restricted space [1]. Plants have been used for therapeutic purposes
for a very long period. A plant, whole or any of its parts, or its extracted substances
that can be exploited for medicinal purposes and producing precursors for producing medicines, is called a medicinal plant [2]. A plant can be called a medicinal
plant if it satises one or more of the following properties: its use in galenical preparations, for example, decoctions, infusions, etc. Its cultivation for the extraction of
pure substances that can be used for direct therapeutic usage or its role in the semisynthesis of a drug. For example, diosgenin, a precursor of various hormones such
as sex hormones, is obtained from Trigonella, Costus species. Any food, spice, or
fragrant plant with medicinal properties, such as Ginger or Turmeric. Any plant that
can be used for manufacturing surgical dressings, such as cotton, jute, or ax [2].
I. Fatima · H. Mukhtar · U. F. Gohar · U. Hameed (*)
Institute of Industrial Biotechnology, Government College University, Lahore, Pakistan
e-mail: uzmahameed@gcu.edu.pk
M. Akram
Government Shalimar Graduate College, Lahore, Pakistan
Institute of Botany, University of the Punjab, Lahore, Pakistan
Z. A. Sajid
Institute of Botany, University of the Punjab, Lahore, Pakistan
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. Zia-Ul-Haq etal. (eds.), Essentials of Medicinal and Aromatic Crops,
https://doi.org/10.1007/978-3-031-35403-8_1
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1.2 Tissue Culture
Plant cells are totipotent and can be turned into new plantlets providing suitable
medium and conditions. In tissue culture, scientists use this ability to grow plant
tissues and organs on articial media in vitro under a precise sterile setting. The
pioneer of tissue culture is German physiologist Gottlieb Haberlandt. In 1902, he
proposed the concept of in vitro cell culture. In his experiment, he cultured the individual palisade cells in knop’s salt solution supplemented with sucrose. The cells
remained alive for one month, accumulated starch, and increased size but failed to
divide. This experiment laid the foundation of plant tissue culture. Thus, Haberlandt
is known as the father of plant tissue culture. New techniques and methods were
developed between the 1940s and the 1960s [3, 4]. Through micropropagation tech-
niques, not only single plants but crops can alsobe grown that are needed to produce
medicinal molecules or extracts [5]. The nutrient medium for plant tissue culture
must contain all necessary nutrients (Fig.1.1) for the growth and development of
the explant [4]. Another important factor that must be considered during tissue culturing is pH.Generally, it is set in the range of 5.4 to 5.8 for both liquid and solid
media [4].
The medium required for the plant invitro micropropagation must contain the
following components [4, 6].
solidifying
agents
Sugars
Fig. 1.1 Essential components of the tissue culture medium
Plant
growth
regulators
Macronutrients
Micronutrients
Plant tissue
culture
medium
Vitamins
Other
organic
supplements
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1.2.1 Macroelements
The elements required in a concentration greater than 0.5mM.L−1 are macroelements. These are always supplemented in the medium, for example, nitrogen, potassium, sulfur, phosphorus, etc. [7].
1.2.2 Microelements
These are also known as trace elements and are required in an amount less than
0.5 mM.L−1 concentration. These nutrients are always employed in the medium.
Examples of micronutrients are iron, boron, nickel, manganese etc. [7].
1.2.3 Sugar
Sugars are the source of carbon and energy in the medium. These are always supplemented in the medium but may be skipped under particular circumstances. Examples
of frequently used sugars in tissue culture medium are glucose, fructose, and
sucrose [7].
1.2.4 Vitamins
Vitamins are essential for the growth and development of invitro plant culture.
These are needed to synthesize several compounds involved in different metabolic
processes in cell growth and differentiation. Examples of vitamins used in invitro
micropropagation of plants are thiamin (B1), pyridoxine (B6) and nicotinic acid [7].
1.2.5 Solidifying Agent
A solidifying agent plays a vital role in the tissue culture medium as it solidies the
medium and provides ground for growth. Depending upon the requirement, the
semisolid or solid-state of the medium can be maintained by changing the concentration of the solidifying agent. Frequently used gelling agents are agar, agarose and
gellan gum [7].
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1.2.6 Amino Acids andNitrogen-Containing Compounds
Amino acids act as the source of nitrogen and raw materials for synthesizing other
compounds required for optimal growth of plant culture. For this purpose, hydrolysates of casein, L-glutamine, L-asparagine, and adenine are commonly used in the
plant tissue culture medium [7].
1.2.7 Undened Supplements
Scientists also tested the effects of natural substances on the growth of invitro cul-
tures. These components contribute to the above-mentioned substances as well as
regulate plant growth. Such undened supplements include coconut milk, yeast
extract, malt extract, ground banana, orange juice, tomato juice, and activated charcoal. Their effects on the culture depend on the plant species and can also have
inhibitory effects on the culture. For example, activated charcoal had an inhibitory
effect on soybean cultures [8].
1.2.8 Buffers
Buffers can be added to maintain the pH of the medium. Buffers are benecial due
to their ability to resist pH changes during the active growth of calli [6].
1.2.9 Plant Growth Hormones
Plant growth hormones or plant growth regulators are the most important components of plant tissue culture media as they regulate growth, induce cell proliferation,
and organize culture development. Generally, “ve main classes of plant growth
hormones are: Auxins, abscisic acid, cytokinins, ethylene, and gibberellins” [9].
Table1.1 shows summary of the components and their role in tissue culture medium.
Preparation of plant tissue culture media is an important task and it should be done
in dedicated and clean area provided with all necessary equipment that should be
regularly maintained [4]. Examples of micropropagation medium for plants are
Murashige and Skoog (MS) medium [10], Gamborg (B5) medium, Linsmaier and
Skoog (LS) medium, Nitsch and Nitsch (NN) medium [4].
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Table 1.1
Major components of tissue culture medium and their role
Component Example Role Reference
Macroelements Nitrogen, potassium,
Microelements Iron, boron, nickel,
Sugar Glucose, fructose, and
Vitamins Thiamin (B
Solidifying agent Agar, agarose and Gellan
Amino acids and
other nitrogen
supplements
Undened
supplements
Buffers Organic acids acting as
Plant growth hormone Auxins, abscisic acid,
sulfur, phosphorus
manganese
sucrose
), pyridoxine
(B
6
gum
L-glutamine,
L-asparagine, and
adenine
Yeast extract, malt
extract, ground banana,
activated charcoal
buffers, a mixture of
KH
cytokinins, ethylene, and
gibberellins
1
) and nicotinic acid
and K2HPO
2PO4
Growth, morphogenesis [6, 11]
Tissue growth [6, 11]
Energy source, carbon source,
osmotic agent
Essential intermediates
metabolic, catalysts
Solidies medium [6]
Sources of nitrogen, raw
materials for synthesizing other
essential compounds
Source of peptides, amino acids,
fatty acids, carbohydrates,
vitamins, plant growth
substances
Stabilize the pH of hydroponic
solutions
4
Inducing the production of roots,
shoots, play important role in
development of calli
[6]
[6]
[6]
[6, 8]
[6]
[6]
1.3 Types ofMicropropagation Methods
Different methods of In vitro micropropagation are available for culturing of plants
(Fig.1.2). These include callus culture, organ culture, single cell culture, suspension
culture, embryo culture, anther culture, pollen culture, somatic embryogenesis, protoplast culture, meristem culture [12, 13]. Various types of micropropagation techniques used are shown in Fig. 1.2. Tissue culture techniques are used for the
production of articial or synthetic seeds that are encapsulated shoot buds, cell
aggregates, somatic embryos or any other meristematic tissue having the potential
to regrow into new plantlets in a suitable medium after the storage conditions.
1.3.1 Callus Culture
Callus is dened as the unorganized mass of proliferative plant cells produced when
an explant is grown on an articial nutrient medium under a controlled sterile environment. Calli differ signicantly in texture, appearance, and growth rate based on
the explant source and nutrient medium constitution. The process of callus formation is called callogenesis. Small explants or sections from the plant organs are
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Plant Tissue Culture Techniques
I. Fatima etal.
Callus
culture
Organ
culture
Single cell
culture
Suspension
culture
Embryo
culture
Anther
culture
Pollen
culture
Somatic
Embryogenesis
Protoplast
culture
Meristem
culture
Explant
culture
Fig. 1.2 Various types of tissue culture techniques
inoculated on solidied medium to initiate callus formation [14]. There are two
steps in callus culture. First, biomass is produced with the growth and multiplication
of calluses, and then biosynthesis of compounds takes place from this biomass.
Selecting an appropriate parent plant is crucial to obtain a good yield because the
commencement of high producing callus and buildup of secondary metabolites is
genotype-dependent [15]. Callus cultures are useful for obtaining commercial products like cosmetic, pharmaceutical, nutritional, coloring agents, avors, fragrances
and animal health-related products. Callus culture is ideal when limited space and
time are available. Rather than growing the whole plant, the same specic metabolite can be obtained just by callus culturing [16]. In callus culture, the explant is rst
sterilized with 0.5% NaOCl for 15min and then washed three times with sterile
water. Another method of sterilization is to apply 0.1% mercuric chloride solution
for 8–10min and washing the explant six to eight times with sterile distilled water.
After this, the explants are cultured into test tubes or Petri plates containing MS
medium supplemented with sugar/carbon source, gelling agent, and plant growth
hormones for callus induction. Sometimes callus can get dark or brown in color. To
prevent this, different antioxidants such as ascorbic acid, polyvinylpyrrolidone
(PVP) and activated charcoal can be addedat 0.5–1.0 mg/L concentration. Petri
dishes or the test tubes are sealed tightly with polyethylene lm and placed in the
incubator or growth culture room at a temperature between 22 and 25°C under a
photoperiod of 16h light/8h darkness. When calli show growth and shoot formation, they are subcultured into fresh media for root growth and acclimatization [17].
Figure1.3 is the graphic representation of the procedure of callus culture [18, 19].
The bioactive compounds of calli are collected mainly at the stationary phase of
their growth cycle because their production increases during this phase. Metabolites
can also be collected at different phase depending on theplant species and callus
properties. The identication and quantication of extracted compounds from calli
are carried out using HPLC, LC-MS, etc. [20]. Explants for callus cultures can be
obtained from different parts of plants. For example, explants in wheat can be
mature or immature embryos [21], shoot apical meristems [22], coleoptile [23] an
anther [24] as shown in Fig.1.4. In other instances, other excellent materials for
callus initiation and regeneration are hypocotyls and root explants.
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Sterilization of
Cutting the
explants in small
sizes
Culturing the
explants in suitable
medium
Subculturing them
into shooting
medium
explants with 0.5%
sodium
hypochlorite for
15 min
Incubating the
cultures for 16 h light
and 8 h darkness at
o
C
22-25
Subculturing to
rooting medium
Under light and shade conditions
Fig. 1.3 Schematic representation of callus culture
Fig. 1.4 Different explants
used for wheat tissue
culturing
rinsing them with
sterile water
Callus formation
Primary
acclimatization
in polycarbonate
•
greenhouse
80 to 90% relative
•
humidity
C
25 ± 2°
•
for 14 days.
•
mature
embryos
Treating the
explants with
Tween 20 for 10
min
Subculturing the
induced explants
into fresh medium
Secondary
acclimatization
in the soil
sand and
manure at 1:1:1
ratio
anthers
shoot apical
meristems
Explants
for
wheat
regeneration
coleoptile
immature
embryos
highest
(
frequency of
)
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1.3.2 Organ Culture
The process of excising the whole or part of an organ or organ primordial (e.g., leaf,
node, internode, shoot, axillary bud, root, or seedling) and using it for in vitro
micropropagation is called organ culture. This technique helps to preserve the structure and function of the organs and retains their characteristics. Organ culturing
provides information about growth, differentiation, and development patterns.
Using appropriate phytohormones, the explant organ can differentiate into complex
structures (e.g., cotyledon, shoot tips, leaf disks, hypocotyls, anther internodes,
stem, roots, or thin cell layers) and then ultimately complete plant [12]. Organ culture can be used for diverse applications, including rapid clonal propagation, preservation of endangered plants, production of valuable metabolites and enzymes
(Fig.1.5) [12, 25, 26].
The rst step in organ culture is the aseptic isolation of tissue. The organ/tissue is
treated with 7X-detergent for 5min and then immersed in a freshly prepared, saturated ltered solution of chlorinated lime for 20min. After this, the explant is washed
with sterile distilled water many times and cut into small pieces of tissue. These
small explants are incubated in the culture medium. This method can be modied
slightly depending on the nature of the explant [27]. Organ culturebenets in maintaining high-class mother plant varieties that amass greater concentration of target
compounds [12]. The schematic diagram of the method is shown in the Fig.1.6.
production of
economically
valuable
chemicals
preservation
of
endangered
species
Fig. 1.5 Applications of organ culture
regeneration
and mass
reproduction
of genetically
modified
fertile clones
Uses of
OrganCulture
rapid clonal
propagation
enzymes
production
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