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17.5 Phytochemical Approach in Chemotaxonomy 341
27 compounds were identified, making up 94.34%; T. plicata contained 31 compounds, representing 94.75%; and
T. plicata “gracialis” showed 30 compounds, comprising
96.36%. Each sample had beyerene and rimuene as the
main parts, along with fenchone, alpha- and beta-thujone, and sabnene. All samples had α-thujone (between
50.14 and 62.12%), β-thujone (ranging from 2.70 to 7.06%),
and fenchone (from 0.17 to 7.06%) as the main ketones.
The total ketone concentration in the oil samples varied
from 54.30 to 69.18%, with T. plicata and T. plicata “gra-
cialis” displaying the highest values at 63.59 and 69.16%,
respectively [58].
The researchers analyzed the metabolic compositions of
11 types of Aster plants by using quadrupole time-of-flight
tandem MS and ultra-high-performance liquid chromatography (UHPLC) with photodiode array detection on sample material that was obtained from above-ground parts. To
identify distinctive chemical markers specific to each species within the Aster genus, a thorough examination was
carried out using a metabolomics database, analyzing 95
representative samples from 11 Aster species. The study
used different methods to analyze the data, like principal
component analysis and cluster analysis. All Aster species
were found to have six phenolic acids and flavonoids,
which suggests that these substances may be common to
the Aster genus. Terpenoid molecules were revealed by
metabolite analysis to be promising chemical markers for
interspecies differentiation. Specifically, the predominant
presence of ent-kaurane-type diterpenoid glycosides was
noted across all Aster species, with the exception of Aster
farreri, which exhibited a higher prevalence of oleananetype pentacyclic triterpenoids. The identification of
Qinghai-Tibetan Plateau’s diterpenoid containing Aster
species marks a significant breakthrough. These compounds have been recognized as valuable chemotaxonomic
indicators due to their low abundance, underscoring their
importance in distinguishing between species [59].
17.5.5 Glycosides
The examination of the substance extracted from the glandular trichomes of Geranium carolinianum (Geraniaceae)
revealed the presence of unique disaccharide derivatives.
Asai et al. [59] notably identified specific compounds
like n-octyl 4-O-isobutyryl-α-L-rhamnopyranosyl-(1→2).
Further exploration unveiled additional compounds: n-octyl
4-O-(2-methylbutyryl)-α-L-rhamnopyranosyl-(1→2)6-O-isobutyryl-β-D-glucopyranoside, n-octyl 4-O-isobutyryl
- α -L-rhamnopyranosyl-(1→ 2)-6-O-isobutyryl
-β-D-glucopyranoside, and n-octyl 4-O-isobutyryl-α-Lrhamnopyranosyl-(1→2)-6-O-(2-methylbutyryl)-β-D-
glucopyranoside. These were identified as caroliniasides A,
B, and C, respectively. These findings significantly enhance
our understanding of the unusual group of SMs known as
n-alkyl glycoside derivatives. These substances are commonly present in the glandular trichome secretions of
Geranium plants and show promising potential for application in chemotaxonomy [60].
Dioscorea species have been traditionally employed in
medicinal practices or as primary constituents for the production of steroid medications, owing to their rich content
of steroidal saponins. HCA was employed to examine the
chemotaxonomy of 12 species (27 taxa) indigenous to
China, with a focus on the diversity of their identified
metabolites. UHPLC coupled with quadrupole time-offlight tandem MS (UHPLC-QTOF-MS/MS) was utilized for
this assessment. Twenty-eight SMs were found, the majority of which were steroidal saponins. The HCA results
showed that Dioscorea bulbifera L. was differentiable for
pennogenin-type steroidal saponins from species belonging
to sect. Stenophora Uline. Dioscorea zingiberensis stands
out among other members of the Stenophora Uline group
due to its manifestation of two unique saponins. This discovery suggests the possibility of reclassifying Dioscorea
banzhuana as a subsection of Stenophora. Furthermore, it
is plausible that Dioscorea nipponica subsp. Rosthornii and
D. collettii var. hypoglauca could be considered distinct species, thus requiring their separation from their original subspecies or varieties [61].
Seven phenylethanoid glycosides, including verbascoside, echinacoside, angoroside A, cistantubuloside B1,
wiedemannioside C, campneoside II, and cistantubuloside
C1, were discovered in two Pedicularis species found in the
Dolomites region. Furthermore, a variety of iridoid glucosides, such as aucubin, euphroside, monomelittoside, mussaenosidic acid, and 8-epiloganic acid, were also identified.
These findings are of considerable taxonomic significance
within the Asteridae family, highlighting the importance of
phenylethanoid glycosides and iridoids in chemotaxonomy. Additionally, Pedicularis verticillata exhibited the
presence of two notably rare constituents within the
Lamiales family, namely excelside B and ligustroside,
which represent unexpected secoiridoids. Certain compounds exclusive to Pedicularis rostratocapitata, namely
8-epiloganic acid, campneoside II, cistantubuloside C1,
ligustroside, and excelside B, as well as those found solely
in P. verticillata, such as angoroside A, cistantubuloside B1,
and wiedemannioside C, could be regarded as distinctive
indicators for these respective plant species [62].
17.5.6 Lignans
Diphenolic substances made up of two phenylpropane units
are called lignans [63]. In examining the fruit composition of
342 17 Comparative Phytochemistry and Chemotaxonomy
nine European Cirsium species, which include members
from the Cephalonoplos, Chamaeleon, and Eriolepis sections, a diverse array of four lignans, three neolignans, and
three sesquineolignans was exhibited. These compounds
play crucial roles as chemotaxonomic indicators. Particularly
noteworthy is the identification of desmethyl balanophonin
and desmethyl picrasmalignan as PMs within the
Chamaeleon section for the first time. Furthermore, previously exclusive to Cirsium eriophorum, the presence of prepi-
crasmalignan and prebalanophonin was observed in Cirsium
boujartii and Cirsium vulgare, underscoring their chemotaxonomic relevance within the Eriolepis section [64].
17.6 Limitations of Chemotaxonomy
The quantitative distribution of SMs in any plant is affected
by numerous factors. The quantitative accumulation and
biosynthesis pathways of SMs are significantly influenced
by ecological factors. Various biotic and abiotic factors have
the potential to influence the biosynthesis pathways of SMs.
Variations in secondary metabolite production can impose
limitations on chemotaxonomic studies. Environmental
stressors such as temperature, humidity, soil quality, and
altitude contribute to differences in secondary metabolite
production among species.
17.7 Conclusion
Medicinal plants hold immense importance in human
existence. However, a notable challenge in their study lies
in their classification. Diverse plant families and species
are known to have varying taxonomic classifications. The
idea of chemotaxonomic classification, which categorizes
medicinal plants based on their chemical traits, provides a
strong and adaptable approach. Chemotaxonomy offers
molecular insights into the diversity of species and their
evolutionary connections, thereby enhancing conventional taxonomic approaches. Chemotaxonomy provides a
thorough and precise classification of species by examining lipids, proteins, nucleic acids, and PMs and SMs.
Developments in analytical methods and integrative strategies keep improving the accuracy and usefulness of
chemotaxonomy across a range of domains, such as medicine development, agriculture, and biodiversity preservation. Chemotaxonomy will be more crucial to the
sustainable use and exploitation of Earth’s biological
resources as our knowledge of the chemical diversity of
life grows.
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18
Medicinal Plant Biotechnology
Anil T. Pawar1, Amol A. Tagalpallewar1, Manasi Mishra2, Arti G. Swami1, Akshay M. Baheti
1
Department of Pharmaceutical Sciences, School of Health Sciences and Technology, Dr. Vishwanath Karad MIT World Peace University, Pune-411038, Maharashtra, India
2
Department of Biosciences and Technology, Dr. Vishwanath Karad MIT World Peace University, Pune-411038, Maharashtra, India
1
18.1 Introduction
Since ancient times, nature has provided a wealth of useful
traditional medicines, many of which are made from plant
sources. Throughout the history, medicinal plants have
been utilized to prevent and treat health aliments, particularly chronic illnesses. New findings related to discovery of
drugs from natural resources have always had a significant
impact on the pharmaceutical industry [1]. According to
the estimates, over 80% of people globally rely mostly on
conventional medications, most of which are herbal [2, 3].
There is a significant and growing need for herbal medicinal products and supplements on a global scale over the
last three decades. Different approaches have been used to
increase the bioactive compounds in medicinal plants. The
generation of secondary metabolites from numerous plant
species is currently one of the major applications of biotechnology. The area of medicinal plant biotechnology is
still growing and can enhance the production of plant
metabolites and products with added value.
The two major sub-domains of plant biotechnology are tissue culture and genetic engineering. The aseptic in-vitro culture of cells, embryos, seeds, tissues, organs, and protoplasts
on nutritional medium is known as tissue culture. The term
“genetic engineering” describes molecular modifications of
organisms that directly affect their DNA. Incorporating
unique features into microbes, plants, and animals is possible
through genetic engineering, which can change the ability of
organisms to synthesize entirely new compounds like hormones, vitamins, vaccines, monoclonal antibodies, etc.
18.2 Plant Tissue Culture
The aseptic (free from microorganisms) in-vitro cultivation
of any portion of the plant (flower, root, leaf, stem, etc.)
under closely supervised environmental and nutritional
conditions is plant tissue culture. These conditions include
medium, pH, growth regulators, temperature, gaseous and
liquid environments, as well as an appropriate supply of
nutrients. The tissue culture process is summarized in
Figure 18.1.
Under controlled conditions, somatic cells (“soma”
means “body”) differentiate into an entire plant. Due to
the plasticity and totipotency of plants, whole plant can be
created from any section of the plant (explants). Plasticity
is the ability of plants to alter their development, growth,
and metabolism in order to survive in and adapt to a particular environment. Since totipotency enables a plant to
retain its genetic potential, any explants utilized in the
process of regenerating new plants will have the same
genetic composition as the parent plant. Dedifferentiation
and redifferentiation of cells are the ways for cells to
exhibit their totipotency. Meristematic cells divide into
two or more types of organs, tissues, or cells that are qualitatively different from one another through a process
known as differentiation. Cells mature through differentiation. The process by which developed cells return to a
meristematic condition in order to produce a callus is
known as dedifferentiation. Redifferentiation is the process by which callus cells can transform into an entire
plant or an organ of a plant [4–6].
348 18 Medicinal Plant Biotechnology
Dedifferentiation
Explant
Plant in soil
Isolated cell
Culture media Callus
Plant regeneration
Shoot formation
Re-dedifferentiation
Figure 18.1 Overview of tissue culture process.
Table 18.1 Major tissue culture-related discoveries.
1901 The word totipotency was first coined by T. H. Morgan to refer to a cell’s potential to develop into an individual plant
1902 German botanist Gottlieb Haberlandt was the first to develop in-vitro cell culture
1904 Hannig propagated embryos of various cruciferous species using mineral salt and sugar solutions.
1908 Simon was able to regenerate a bulky callus, buds, and roots from a poplar stem segment.
1922 Root and stem tips were successfully cultivated by Kotte from Germany and Robbins from the USA, respectively.
1926 Indole acetic acid was the first plant growth hormone discovered by Fritz Went.
1934 An indefinite culture of tomato roots demonstrated by P. R. White.
1935 Indole acetic acid enhanced cambial activity, as demonstrated by Snow.
1937 Yeast extract was replaced with three B vitamins—thiamine, pyridoxine, and nicotinic acid—as a growth supplement in
tissue culture medium by White.
1939 Gautheret, White, and Nobecourt created an infinite number of callus cultures using an auxin-enriched media.
1941 In order to promote cell division in datura, Overbeek et al. were the first to use coconut milk.
1946 Ernest Ball used shoot tip culture to grow complete lupinus plants.
1954 The first person to separate callus tissues into individual cells was Muir. He gave an example of how callus tissues split
into a single cell when they are placed in a liquid medium and shaken.
1955 An adenine derivative known as kinetin was isolated by Skoog and Miller from autoclaved yeast extract.
1957 Skoog and Miller introduced the idea that hormones (auxin: cytokinin) regulate the creation of organs.
1959 Daucus carota callus clumps and cell suspension were used by Reinert and Steward to regenerate embryos.
1960 Edward Cocking was the first to use enzymatic cell wall breakdown to isolate protoplasts.
1960 Bergmann separated individual cells from the cell suspension. This process is known as the plating procedure.
1962 Murashige and Skoog (MS) medium with a higher proportion of salt was discovered by Murashige and Skoog.
1962 Test tube fertilization technology was developedby Kanta and Maheshwari.
1963 Letham isolated a substance with kinetin-like properties from young maize endosperm named zeatin.
1966 Steward developed carrot plants from a single cell to demonstrate totipotency.
1966 Using Datura pollen grains, Guha and Maheshwari produced the first haploid plants.
1970 The first restriction enzyme from Haemophilus influenza (HindIII) was discovered by Smith and Nathans.
1970 Reverse transcriptase from the RNA tumor virus was identified by Baltimore.
1972 Through protoplast fusion, Carlson created the first inter-specific hybrid of Nicotiana (N. glauca and N. langsdorfii).
1972 Berg created the first recombinant DNA by fusing the λ and SV40 viruses.
1974 Zaenen et al discovered Ti plasmid is tumour inducing principle of agrobacterium
1975 O’Farrel developed a two-dimensional gel electrophoresis technology with high resolution.
1977 Agrobacterium tumefaciens’ Ti plasmid DNA was successfully incorporated into plants by Chilton and coworkers.
1978 Melchers and coworkers used somatic hybridization to cross potato and tomato to create pomato.
18.2 Plant Tissue Culture 349
1980 Zambryski detailed the structure of T-DNA and border sequences.
1981 Coining of the term somaclonal variation by Larkin and Scowcroft.
1983 The Polymerase Chain Reaction (PCR), which amplifies DNA, was created by Kary Mullis.
1984 By using Agrobacterium to alter tobacco, Horsh et al. generated transgenic tobacco.
1987 For plant transformation, Klien et al. developed a biolistic gene transfer technique.
1988 Through electroporation, Mettler, I. J., Rhodes, C.A., Detmer, J. J., Pierce, D.A., and Mascarenhas, D created a transgenic
1988 Through electroporation, Toriyama, K., Hinata, K., Uchimiya, H., and Arimoto, Y. created transgenic rice plants.
1989 Shimamoto, K., Terada, R., Izawa, T. and Fujimoto, H produced fertile transgenic rice plants regenerated from
1991 Fodor developed the DNA microarray technology.
1995 Fleischmann and coworkers sequenced Haemophilus influenza.
1997 The Escherichia coli (E. coli) genome was sequenced by Blattner and coworkers.
2000 Ingo Potrykus developed Golden Rice.
maize plant.
transformed protoplasts.
18.2.1 History of Plant Cell Culture Technology
It was from the decorticated elm tree when Henri-Louis
Duhamel du Monceau discovered callus development in
1756. The discovery of plant tissue culture was made possible by this very ancient experiment. Schleiden and
Schwann first put forth the hypothesis of totipotency in
1838. They suggested that because each cell has the capacity for autonomy, under ideal circumstances, every cell
should be able to grow back into a whole plant. The proof
of totipotency in the lab conditions used by Schleiden and
Schwann was unsuccessful. German botanist Gottlieb
Haberlandt made the first effort in 1902 to cultivate isolated single palisade cells in Knop’s salt solution enhanced
with sucrose. The cells continued to grow in size for a
month while remaining alive, but they did not divide.
Although he did not succeed, he did lay the groundwork
for tissue culture, earning him the title of “the father of
plant tissue culture.” Following that, several tissue culturerelated major discoveries happened which are highlighted
in Table 18.1 [7, 8].
18.2.2 Nutritional Requirements and Cultural Media
The soil and the environment provide vital nutrients to
plants that flourish in the wild. The root system of plants
absorbs the inorganic nutrients from the soil along with
water, in the form of ions. Carbon dioxide from the atmosphere is used via photosynthesis to produce energy.
Minerals and fixed carbon help plants to synthesize several other essential compounds like vitamins and plant
growth regulators via metabolic pathways [9]. In-vitro
plant tissue culture requires all the nutrients, just like
plants do in the wild. Plant tissues and organs in-vitro cul-
tivation occur on an artificial culture medium supplemented with nutrients that stimulate growth. For the
growth of plant tissue, various researchers (including
White, Murashinge and Skoog, Schenk and Hildebrandt,
etc.) have occasionally proposed the components of a culture media. The medium’s inorganic and organic chemical
additives should be precisely determined to- (i) give the
plant tissues, cells, and organs in culture the nourishment
they require to survive, and (ii) maintain the ideal physical
conditions of osmotic pressure, pH, etc. No single medium
can ensure optimal growth of the plant tissue since nutritional needs vary from species to species. The most suitable medium for a particular tissue of a species must be
determined by the trial-and-error method using the following components:
i) Inorganic nutrients: Micronutrients and macronutrients
ii) Carbon and energy source
iii) Organic supplements: Vitamins and amino acids
iv) Solidifying or gelling agent
v) Growth regulators
(i) Inorganic Nutrients
The inorganic nutrients needed by a plant cell culture are
the same as what natural plants need. Each nutrient has a
different optimal concentration for maximizing growth
rates. The International Association for Plant Physiology
defines macronutrients as elements with concentrations
larger than 0.5 m and micronutrients as those with concentrations less than 0.5 m [10, 11].
350 18 Medicinal Plant Biotechnology
1. Macronutrients
Phosphorus (P), calcium (Ca), magnesium (Mg), sulfur (S),
nitrogen (N), and potassium (K) are the main components.
They are used as salts in plant culture media. Salts separate
into cations and anions in weak aqueous solutions. Thus,
plant cells absorb magnesium, calcium, and potassium as
2+
the cations Mg
(NO
) form of nitrogen is primarily absorbed; however,
3
uptake of ammonium (the cation, NH
the phosphate ions H
sulfate ion SO
, Ca2+, and K+, respectively; the nitrate
+
); phosphorus as
4
2PO4
2
- may also occur.
4
and HPO
2
; and sulfur as the
4
Nitrogen: Nitrogen is an element that is found in chloro-
phyll, protein, and nucleic acids. For cultured tissues to
develop and differentiate, nitrogen is essential. Between 25
and 60 m is the range of inorganic nitrogen. Mostly,
nitrate and ammonium compounds are used to supply
nitrogen in the culture media. Even if nitrates are given to
the media, they first need to be converted into ammonium
before plant cells can use them. However, ammonium can
not be added directly into to the medium as a high concentration of ammonium ions may be hazardous to plant cells
and a medium’s acidification may result from the uptake
of ammonium ions. It is advantageous to combine nitrate
and ammonium in the medium. Nitrate by itself causes
the medium’s pH to drift toward basicity; however, the
addition of ammonium compounds to nitrate stops this
from happening. It is crucial to consider the medium’s
nitrate to ammonium content. Ammonium is often
administered at concentrations of 2–20 m, and nitrate is
typically added at quantities of 40 m. Magnolia “yellow
bird” grew most abundantly (as a shoot culture) at 7.43 m
and 6.25 m NH
NO
3
at 25.04 m NO
3
+
, while culture mortality occurred
4
[12].
Potassium: The main positive ion that plants use to counter-
act the negative ion is potassium. The ideal amount of
potassium is 20 m. The movement of potassium ions
through cell membranes occurs very quickly. In addition
to stabilizing the cell’s pH and osmotic potential, they neutralize organic anions produced in the cytoplasm. For several proteins and enzymes, potassium serves as a cofactor.
Proteins show high specificity for potassium and become
active in the presence of potassium. A deficiency of potassium is said to decrease the rate of phosphate absorption
and also lead to hyperhydricity (Pasqualetto et al., 1988).
Hyperhydricity is a physiological disorder that causes a
reduction of propagation and death of tissues because of
stressful conditions by waterlogging [13–14].
Calcium: Although calcium is a significant cation and
aids in the balance of anions in plants, it is less mobile
than potassium and magnesium. The middle lamella of
cell walls, the physiological properties and structure of
cell membranes are affected by the calcium. Calcium is
provided as calcium chloride or calcium nitrate, with a
3 m concentration being optimal. Many plant enzymes
require calcium, and calcium is a cofactor in the
enzymes required for hydrolysis of ATP. Many of the
responses brought on by plant growth agents require
+2
the Ca
ion, which is involved in in-vitro morphogenesis. Proteins and phospholipids must be deposited on or
within plasma membranes, and calcium is necessary for
this. Lack of calcium in plants causes poor root development, blackening and curling of the apical leaf margins,
and death of the shoot tip [15].
Phosphorous: Phosphorus contributes to the structure of
nucleic acids. It is also found in substances that are
involved in energy transfer, nucleic acid synthesis, and
protein synthesis. Phosphorus is added into the culture
medium as phosphate in sodium and potassium hydrogen
phosphate at a concentration of 1.1–2.0 m. Phosphorus
is absorbed into plants by an active process. The growth of
tissues is frequently hampered by phosphorus concentrations higher than 2 m [16].
Magnesium: A crucial part of the chlorophyll molecule is
magnesium. The center atom of the chlorin ring of chlorophyll is magnesium. Similar to potassium, magnesium
is mobile, readily diffuses throughout the plant, and
neutralizes negatively charged ions. Magnesium is
mostly provided as magnesium sulfate, which is present
in concentrations of 1–3 m [17].
Sulfur: Sulfur is provided in quantities ranging from 1 to
2
3 m as the SO
ion. Sulfur plays a significant role in
4
protein synthesis and is a component of various amino
acids, including methionine and cysteine. It is a component of the vitamins biotin and thiamine and stimulates
specific enzyme systems. Plants utilize sulfur in the formation of lipids. Sulfur deficiency affects the quantity of
chlorophyll in plants and inhibits protein formation [18].
2. Micronutrients
The microelements usually consist of boron (B), manganese
(Mn), iron (Fe), zinc (Zn), copper (Cu), chloride (Cl), molybdenum (Mo), cobalt (Co), and iodine (I); however, other elements like nickel (Ni), silicon (Si), and aluminum (Al)
frequently occur in various culture medium [19-21]. Though
required in trace amounts, these are also equally essential
for optimal plant growth depending on specific species.
Manganese: Manganese is added to the medium at a con-
centration of 5–30 as manganese sulfate. Manganese
serves as a cofactor in the processes of photosynthesis and
respiration. Decarboxylases, dehydrogenases, kinases,
and numerous other enzymes need it for their optimal
function [22].