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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5714_Библиотеки_им_академика_М_И_Перельмана.pdf
GENETICS AS APPLIED TO MEDICINAL HERBS
Gregor J. Mendel (1865) showed that certain hereditary factors operate in all biological species.
The Danish biologist Wilhelm Johannsen (1909) named these factors as genes. The genes not only
transmit hereditary characters but also mastermind the entire process of life. The genes are located
in chromosomes that are themselves situated in the nucleus of the cell. It is said that the genes form
the riddle and the chromosomes represent the mystery. Much of the mystery surrounding the genes
was cleared up with the discovery of the structure of the DNA by J.D. Watson and Francis Crick in
April 1953. The structure of the DNA resembles a long-rope ladder twisted around like a corkscrew.
The two sides of the ladder are long chains of sugars and phosphates in repeated sequence
(Fig. 6.1). DNA contains the bases, namely, adenine (A), cytosine (C)
,
thymine (T) and guanine (G), that
make up the genetic code that is not only complex but also extensive. In 1977
,
Fred Sangar pointed out
that the DNA code of a virus, when decoded by computer, come to a printout of 15 metres.
The genes control all functions of cell and body growth. The two main events in the life of most
cells are multiplication (by division) and synthesis of proteins. Both these operations are carried out
on the basis of blueprints coded in the genes.
The science of genetics has made valuable contributions to the improvement of plants with
economic, medicinal and ornamental values. This improvisation is attained by selective breeding,
mutation, polyploidy and hybridization. It has important roles in mitosis and meiosis of the cells.
Medicinal Plant Biotechnology
6
CHAPTER
Genetics as Applied to Medicinal Herbs
Mutation
Polyploidy
Chemodemes (Chemical Races)
Hybridization
Genetic Engineering and Recombinant
DNA Technology in Plants
Plant Tissue Cultures as Sources of
Biomedicinal Compounds
Historical Development
Types of Cultures
Culture Medium
Surface Sterilization of Explants
Establishment of Cultures
Phytopharmaceuticals in Plant
Tissue Cultures
Bioproduction of Useful Metabolites
in Hairy Root and Multiple Shoot Cultures
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76
Mitosis results in precise equal distribution of chromosomes from a parent nucleus to the daughter
nuclei. Further, it maintains equilibrium in the amount of DNA contents of cells. Mitosis is usually
associated with vegetative propagation of plants. The somatic number is the number of chromosomes
in plant cell nucleus, which in normal conditions remains constant, but varies with different species
(Fig. 6.2).
Meiosis occurs in reproductive cells of sexually reproducing species. It has a genetic significance
because of formation of four haploid nuclei from a single diploid one, in two successive divisions;
thus, balancing the doubling of chromosome numbers that results from fertilization. In meiosis,
the crossing over provides new combinations of genetical substance. Hence, new combinations of
characters in offsprings occur. The process of segregation of chromosomes also occurs in meiosis.
A
A
A
A
A
A
A
A
A
C
C
C
C
C
C
CG
G
G
G
G
G
T
T
T
T
T
Figure 6.1 Structure of DNA.
Medicinal Plant Biotechnology
77
These techniques of genetic manipulations in plant cells have made much impact to improve
yield of medicinal plants.
MUTATION
Mutation is represented as variation in characters of the species. It is caused either due to environmental
changes or changes in hereditary constitution. Normally, as a response to environmental changes, the
variations are observed, but the original traits are restored when changes in environment disappear or
they are withdrawn. This type of change and restorage is not heritable and termed only as phenotypic
variations. It is evident that between organisms of similar genotype, there are phenotypic variations.
But when a change occurs in the genome of an individual that is not caused due to environment, it
may make a permanent evolutionary change. This is termed as mutation that represents a sudden
change in a genotype causing qualitative or quantitative alterations of genetic material. Mutations
are again distinguished into two types, such as chromosomal mutations and point mutations. The
chromosomal mutation is also called chromosomal aberration that leads in most cases to changes
in amount or position of genetic material. On the other hand, the changes with a gene or cistron of
DNA molecule cause point mutations, and they are permanent and heritable (Fig. 6.3).
Mutation that occurs due to some unknown reason from nature is known as spontaneous
mutation.
This has been observed in some plants, bacteria and viruses. Mutations can also be
induced by artificial means ( induced mutation) with certain reagents called mutagens. Exposure of
cells to UV rays, X-rays, ionizing radiations and abnormal environment may induce mutation. The
chemical mutagens commonly used are nitrogen mustard, formaldehyde, nitrous acid, ethyl ethane
sulphonate, 5-bromouracil, 2-aminopurine, manganese chloride, etc.
DNA replicates
Mitosis
Parent cell
2 Daughter
cells
Parent cell
DNA replicates
2 Daughter
cells
4 Daughter
cells
Meiosis
Figure 6.2 Mitosis and meiosis.
Textbook of Pharmaceutical Biotechnology
78
First round of
replication
Mutation
Mutant strand
replication
Mutation
fixed
Second round of
replication
Wild-type
strand replication
ACA
TGT
ATA
TGT
ATA
T
A T
ACA
TGT
ACA
TGT
ACA
TGT
Figure 6.3 Mutation.
Mutation
Isolation
Founder’s
principle
Polyploidy
Hybridization
Genetic pool
Natural
selection
Reunion
Species
formation
Figure 6.4 Effect of various factors on species formation.
The mutations induce morphological and anatomical changes as well as changes in the
chemical composition of medicinal and aromatic plants. In some cases, favourable changes and
yields in active constituents of plants have been achieved. Mutations may give rise to building
the resistance of a medicinal plant towards certain disease. But, in all these cases, the plant may
become susceptible to climatic conditions, certain other diseases, retardation in growth, etc. These
undesirable effects are to be eliminated by breeding and selection (Fig. 6.4).
Medicinal Plant Biotechnology
79
Artił cial Mutation
Artificial mutations are induced by artificial means in the living organism by exposing them to abnormal
environment. These include radiations, certain physical conditions such as temperature and chemicals.
The discovery of chemodemes has enabled bioscientists to select high-yielding chemical strains or
to eliminate chemical strains that contain toxic principles. The chemodemes are induced by breeding in
some species, so as to manipulate active constituents with a view to enhance the therapeutic efficacy.
Radiation Mutations
The electromagnetic waves of short wavelength (ultraviolet light, X-rays, gamma rays, alpha and
beta rays) are radiation mutagens. The X-rays and gamma rays are called ionizing radiations, and
also include alpha particles, beta rays, thermal and fast neutrons. Due to ionizing radiations, in
many cases, water molecule in a biological system releases one electron and becomes unstable and
eventually splits into hydrogen ion and hydroxy radical. Hydrogen ion reacts with O
2
and produces
hydroperoxyl (HO
2
) radical. Both these radicals, such as hydroperoxyl and hydroxy are potent
oxidizing agents. When chromosomes and their DNA are struck by such radicals, they react due to
which sugar phosphate part of DNA may be impaired leading to chromosomal mutations, such as
deletions, additions, breaks, inversions and translocations.
Chemical Mutations
Some chemical mutagens, like nitrogen mustard, formaldehyde, nitrous acid and ethyl ethane
sulphonate alter chemical constitution of DNA bases and cause transitional substitution in DNA. But,
some compounds like 2-aminopurine, urethane, 5-bromouracil, caffeine, triazine, phenol and certain
other carcinogens act as a base analogue and bring out certain error mutations in DNA. In general,
the chemical mutagens have profound cellular effects like production of abnormal DNA (e.g. nitrogen
mustard), inhibition of deoxyribonucleotide synthesize (e.g. deoxyadenosine) and inhibition of
cytochrome oxidase with resultant peroxide formation (e.g. inorganic cyanides). The artificial production
of mutations is an important milestone in the development of cultivation technology of medicinal plants.
The higher content of solasodine is obtained by applying radiation and chemical mutagens in Solanum
khasianum. The chemical mutagens also increase morphine content in Papaver somniferum. The yield
of tuber of Dioscorea bulbifera and its diosgenin content is increased by radiation. The medicinally
important characters of Atropa belladonna have been enhanced by radiations and chemical mutagens.
The capsicum seeds ( Capsicum annuum) treated with sodium azide and ethyl methane sulphonate have
led to plants with higher contents of capsaicin. The agronomical performance in terms of harvest index of
Mentha arvensis var. piperascens ( Japanese mint) has been improved by exposure to gamma radiations.
The positive effects of artificial mutations have been witnessed on different species of Mentha.
POLYPLOIDY
The induction of polyploidy has useful effects on medicinal plants, such as digitalis, mentha,
datura, hyoscyamus, belladonna, lobelia, etc. The specific number of chromosomes is a character
Textbook of Pharmaceutical Biotechnology
80
of each species and is called genome. Euploidy is a type of ploidy in which genome contains whole
set of chromosomes. Euploidy includes monoploidy, diploidy and polyploidy. When the organism
contains more than two sets of chromosomes ( genomes), it is called polyploidy. The polyploidy
occurs in a multiple series of the basic chromosome or genome numbers of 3, 4, 5, 6, 7, 8, etc. and
accordingly, it is called triploidy, tetraploidy, pentaploidy, hexaploidy, heptaploidy and octaploidy,
respectively (Fig. 6.5).
Polyploidy is caused by physical agents like X-rays, centrifugation, temperature chocks
and chemical agents mainly, colchicine, veratrine, sulphanilamide, hexachlorocyclohexane and
mercuric chloride. The chemical agents cause disturbance to mitotic spindle of dividing diploid
cell and induce nonsegregation of already duplicated chromosome and thus, convert diploids into
tetraploid cells. It may cause formation of new species, adaptability to various habitats and mainly
accumulation of vitamins.
The ideal chemical agent to induce polyploidy is colchicine, an alkaloid obtained from
Colchicum autumnale, Colchicum luteum, Colchicum speciosum and other speices of Colchicum.
The tropolone alkaloid, colchicine prevents sister chromatids from separating into daughter nuclei
at anaphase. These chromatids remain attached by their common centromere in C-metaphase. The
chromatids eventually separate, but remain in the same nucleus. An interphase occurs, followed
by a second C-metaphase, involving a doubled chromosome complement. Hence, the chromatid
pairs are doubled in second C-metaphase. Likewise, the cell undergoes one, two or more than two
rounds of DNA replication and results into polyploidy. The activity of colchicine is caused due to
its interaction with disulphide bonds of spindle protein and by inhibition of conversion of globular
proteins into fibrous proteins. The capacity of colchicine to induce polyploidy varies along with
its different derivatives. Colchicine treatment to medicinal plants has given promising results in
many cases. Chemically induced mutations may lead to variations in biochemical composition of
plant. In stramonium, the yield of crop is enhanced by 60–150% in 4n form. Plants such as lobelia,
cinchona, belladonna, squill, cannabis, acorus and poppy also show increased yield of respective
compound in 4n form. Polyploidy may cause a reduction in total glycoside contents of Digitalis
purpurea and Digitalis lanata, but in the later, it raises slightly the contents of lanatosides A and
B. The increase in chemical contents may not be coincidental with phenotype of medicinal plant.
4 Single
chromosomes
4 Doubled
chromosomes
4 Doublets Chromatids
separate and
spindle dissolves
8 Single
chromosomes
Prophase Metaphase Anaphase
Figure 6.5 Polyploidy.
Medicinal Plant Biotechnology
81
There may be reduction in size in addition to enhancement in the content of active constituents.
Some plants do not show any change in chemical contents as a response to polyploidy.
CHEMODEMES (CHEMICAL RACES)
Chemodemes
are regarded as a group of plant species having identical morphological characters,
differing in their chemical nature. Chemodemes are, therefore, considered as chemically separate
groups within species. The chemodemes can be established only by growing different plants of
a species in identical conditions preferably, from the seeds and for many generations. By this
way, it shows variations either in type or contents of certain constituents, including secondary
metabolites of medicinal importance. The chemical characters acquired as a result of chemodemes
are hereditary.
Chemical races have been reported in D. purpurea and D. lanata. These chemical races in D.
purpurea yield different proportions of glycosides obtained from digitoxin and gitoxin. Based on
lanatosides A and C contents, the chemical races in D. lanata are D. lanata Ehrh., chemovarieties
A and C. The chemodemes in Rheum palmatum exhibit different rhein–chrysophanol ratio. The
different varieties of Prunus communis (almond) differ by the presence or absence of amygdalin.
Chemical races have also been reported in Duboisia myoporoides, Duboisia leichhardtii and
Claviceps purpurea. Three chemotypes are evident in Withania somnifera. The chemotype I
contains mainly withaferin; chemotype II has identical compounds, whereas chemotype III contains
withanolides (a mixture of steroidal lactones).
The volatile oil-bearing plants like Cinnamomum zeylanicum, Cinnamomum camphora,
Eucalyptus dives, Ocimum menthaefolium and Ocimum sanctum exhibit chemical races, with
varied contents of aromatic constituents.
HYBRIDIZATION
A hybrid is an organism that results from crossing of two species or varieties differing at least in
one set of characters. The process through which hybrids are produced is called hybridization.
The resultant hybrids are monohybrids (one pair of different characters), dihybrids (two pairs of
different characters) or polyhybrid (more than two pairs of different characters). The process of
hybridization helps in yielding a single variety with favourable characters of other varieties or
species and some times, producing new and favourable characters that are not present in both the
parents (Fig. 6.6).
The hybridization of Withania somnifera—Israeli chemotype II and W. somnifera—South
African chemotype have led to the formation of a new hybrid that contains three new withanolides.
The hybrids such as D. purpurea u D. lanata and D. purpurea u D. lutea contain lanatoside A as
main glycoside, along with lanatosides B and E. However, the hybrids do not contain lanatoside
Textbook of Pharmaceutical Biotechnology
82
C or purpurea glycoside A. During the cross of Solanum incanum (1.8% solasodine) and Solanum
melongena (traces of solasodine), first generation bears more fruits (berries) with solasodine content
up to 0.5%. However, the second generation has proved to be a high-yielding source of solasodine.
A recent development in hybridization is the protoplast culture. The plant protoplasts are the cells
without cell walls. Such protoplasts in cultures can be fused together ( protoplast fusion or asexual
hybridization). The fusion can be arranged in cells of same origin or between different species.
GENETIC ENGINEERING AND RECOMBINANT DNA
TECHNOLOGY IN PLANTS
The technique of artificial synthesis of new genes and their subsequent transplantation in the genome
of an organism or the method of correcting defective genes of organism by molecular biological
techniques form the discipline known as genetic engineering. In recent years, these techniques
have gained importance in relation to their utility in drugs and pharmaceuticals. The secondary
metabolites from plants that are expected to give promising results by genetic engineering include,
cardiac glycosides, morphine alkaloid, stigmasterol or diosgenin (for synthesis of steroid hormones
and corticosteroids). In respect of such plants, the transfer of entire biosynthesis route to bacteria by
technique of gene technology is expected to yield different economic processes (Fig. 6.7).
Recombinant DNA technology involves gene splitting, so as to change characters of plants and
animals by implanting in them genes from other organisms and, in some cases, even from other
species. Important genetically engineered products available in market are human insulin, human
growth hormone, interferons, hepatitis B vaccine, interleukin-2 (to fight certain types of cancer),
`-endorphins (for treatment of pains), tissue plasminogen activator (TPA; for treating heart attack),
somatostatin (for treatment of pituitary diseases) and erythropoietin or EPO (for treating anaemia in
patients undergoing kidney dialysis).
Inbred + Inbred = Hybrid
Figure 6.6 Hybridization.
Medicinal Plant Biotechnology
83
Transgenic Plants
Application of genetic engineering techniques for the improvement of plants is a fast-developing
area of research. The advent of modern technique of genetic modification has enabled to remove
individual genes from one species and insert them into another, without the need for sexual
compatibility. Once the new gene has been inserted into the plant, offspring that contains the copies
of the new gene can be produced in the traditional manner.
Transgenic plants (Fig. 6.8) also carry the stable integrated foreign genes. The introduction
of foreign gene is made by genetic transformation ( gene transfer), also called transfection. It is
necessary that DNA remains stable and can pass the new gene to its offspring. Transgenic plants
are useful in causing crop improvement. The herbicide- and insect-resistant species and species
with high yields of oil and protein, as well as other enhanced quantities of products (Fig. 6.9), are
obtained through this technique.
The complete process of transformation includes the following steps:
1. Gene transfer
2. Selection of transgenic plants
3. Recovery of transgenic whole plants
Nucleoid
Ti plasmid
Agrobacterium
tumefaciens
DNA with gene
Site of enzyme
cutting Ti plasmid
T-DNA
Enzymes
Recombinant Ti plasmid
1
Regeneration
of plant
Plant cells in culture
T-DNA integrates
with chromosomes
2
3
Figure 6.7 Recombinant DNA technology in plants.
Textbook of Pharmaceutical Biotechnology
84
Gene Transfer
1. By using Agrobacterium tumefaciens: This Gram-negative bacterium commonly occurs
in soil and causes crown gall disease (tumorous outgrowth) in higher plants. It enters the
plant through the mechanically wounded tissue site. Agrobacterium tumefaciens exhibits
capability to transfer some of its DNA into nuclear DNA of an infected host plant mainly,
because of functions encoded on its tumour-inducing (Ti) plasmid. The transferred DNA or
xDNA of this plasmid encodes substances and acts as plant hormone, causing proliferation of
Undifferentiated
mass of cells
Cells grown in
liquid culture
Mature plant
(resistant)
Growth of
plant
Redifferentiation of
cells into embryos
Herbicide,
cells with DNA
Culture,
bacteria with
Ti plasmid
Mature plant
(suspectable)
Figure 6.8 Transgenic plant.
Figure 6.9 Process of transformation.
Gene transfer or DNA delivery systems
1 Using Agrobacterium tumefaciens
2 Using viral vectors
1 Electrical methods (electroporation)
2 Physical methods microin ection, silicon carbide
whis ers, micropro ectile bombardment, biolistic gene
transfer, sonication)
3 Chemical methods (using polyethylene glycol (PEG),
fusion of protoplasts with liposomes)
Vector-mediated Vector-free