PLANT CELL BIOTECHNOLOGY: RECENT APPROACHES FOR SECONDARY METABOLITE PRODUCTION

CELL BIOTECHNOLOGY
PLANT CELL CULTURE
Recent approaches and Development for production of
secondary metabolites.

1. Biotechnology
• Plant Cell Biotechnology
• General terms
2. Plant Cell Culture
3. Development in Plant Cell Culture
4. Recent Approaches for High Production of secondary
metabolites ( Drugs)
CONTENTS:
2

The definition of biotechnology usually includes the development of methods by
which biological processes may be controlled such that their rate of production
enables economic industrial production (phytoproduction), or by which living
material is obtained that can be utilized in industry, agriculture and forestry, as well
as in gardening and breeding (plant breeding).
3
PLANT CELL BIOTECHNOLOGY:
BIOTECHNOLOGY:
Plant Cell Biotechnology is broadly concerned with experimental research on
plants and plant like organisms (fungi and cyanobacteria).(Stafford, Morris, &
Fowler, 1986)

GENERAL TERMS:
4
Cultures of plant cells taken from their natural
environment and placed under controlled
conditions. Callus a more or less loose
association of cells without visible morphological
differentiation. Cell suspension denotes cultures
consisting of single cells or the smallest cellular
association without differentiation, submersed in
a turbulent medium. Protoplasts are naked cells
of varied origin without cell walls, which are
cultivated in liquid as well as on solid media
Callus, Cell Suspension
and Protoplast: Each cell contains every single attribute
which appears in the differentiated plant.
• Morphological totipotency was
achieved by the regeneration of
completely normal differentiated plants
from callus and protoplasts
• Chemical totipotency was brought by
the examination of anthraquinone
accumulation in the calli of different
parts (root, leaf, shoot, fruit) of Morinda
citrifolia .
Totipotency:
Sterile organs or pieces of tissue used to gain
dedifferentiated cells by proliferation at
sectional planes or wounded areas are called
explants.
These Explants are placed in specific solid
culture media which, because of their
phytohormonal content, encourage cell
proliferation.
Explant:
As a rule, cytokinins and auxins are used as
phytohormones. 2,4-Dichlor-phenoxyacetic acid is
the most powerful, and is therefore called the
dedifferentiation hormone.
Phytohormones: Cytokinins Auxins
6-Benzyl-aminopurine (6-BA) Indole-3-acetic acid (IAA)
6-Furfuryl-aminopurine (kinetin) I-Naphthalene acetic acid (NAA)
2,4-Dichlorphenoxyacetic acid
(2,4-D)

Callus Formation: Usually, the ratio between auxin and cytokinin concentrations
determines whether a culture grows in a disorganized fashion as callus or develops
shoots or roots. A particularly effective agent for callus formation is 2,4-di-
chlorophenoxyacetic acid, generally characterized by its great effectiveness. Its effect
is usually increased by the addition of cytokinin.
Organogenesis: By reversing the conditions suitable for callus formation, the
meristematic cells or groups of cells developing in the course of callus formation are
stimulated to organogenesis. In many dicotyledonous callus cultures, bud formation
is generally initiated by a ratio of 100/10, while callus development is favored by the
inverse ratio 10/100. The relevant ratios in tobacco are different, but they do
demonstrate the dependence of the effect on the donor material. However, the
relative effectiveness of different auxins must be kept in mind. In some cases,
culturing without one of the two hormones is sufficient to induce organogenesis.
5
AUXIN TO CYTOKININ RATIO:

GENERAL TERMS:
7
• Soma clonal variations can be
obtained by subcultring the callus
into different particular stress
environment either by induced
mutation using mutagens or by
subculturing.
• They are clones but with
phenotypic and genotypic
variations.
Soma clonal variations:
• Somatic embryogenesis is an artificial
process in which a plant or embryo is
derived from a single somatic cell.
• Desired product can be obtained from plant
by somatic embryogenesis.
• Through somatic emryogenesis we get
genetically uniform cell.
• Bipolar in nature ( whole plant can grow)
• Disease free large scale production
• Synthatic seed production
• E.g; Somatic culture of tobacco plant to
extract nicotine.
Somatic embryogenesis:
• Morphogenesis (from the Greek
morphê shape and genesis creation,
literally "the generation of form") is the
biological process that causes a cell,
tissue or organism to develop its
shape.
• Organogenesis is the differenciation
of cells into organ system. In
monopolar growth either root or shoot
is grown at a time.
Morphogenesis:

SOMATIC EMBRYOGENESIS:
8
40-60%
Average Profit
Margin
$175,000
Average Annual
Revenue per Doctor
3%

METHODS OF PROPAGATION:
* According to a Survey
9
40-60%
Average Profit
Margin
$175,000
Average Annual
Revenue per Doctor
3%
Market Growth Per
Year

Cell suspension cultures are usually inoculated with the help of a callus fragment
put into a liquid medium. The minimal amount needed is about 2 to 3 g/l00 ml
medium.
10
PROTOPLAST CULTURE:
CELL SUSPENSION CULTURE :
1. Physical Methods
• Grinding with glass beads
2. Enzymatic Methods
• Cellulose
• Hemicellulose
• Pectinase
• The enzyme incubation can be carried out either successively in separate
enzyme solutions or in a mixture of these enzymes for a long time (5-8 h) at low
temperatures (8-25 °C), or alternatively for a short period ( < 2 h) at high

11
GENERAL CULTURE TECHNIQUES :
1. Sterlization
• Sterlization of plant tissue by sodium hypochlorite, bezalkonium chloride, bromine
water.
• Sterlization of nutrient solution by autoclaving, tyndallization or sterile filtered.
• Sterlization of glassware and tools
• Air sterilization
2. Composition of media:
• Inorganic components
• Macronutrients
• Organic components
• Vitamins
• Growth regulators

12
TISSUE CULTURE IS WIDELY USED IN:
PLANT TISSUE CULTURE:
• Plant tissue culture is a set of techniques for aseptic culture of cells, tissues, organs and
their components under desired physical and chemical conditions in vitro and controlled
conditions.
• It explore conditions that promote cell division and genetic reprogramming in vitro
conditions
• It is an important tool in basic and applied sciences as well as in commercial applications.
• Obtaining disease free plant.
• Rapid propagation of plants those that are difficult to propagate.
• Somatic hybridization.
• Genetics improvement of commercial plants e.g; transgenic plants
• Obtaining androgenic and gynogenic haploid plants for breeding programs.

13
HISTORY:
• Gottlieb Haberlant justifiably is known as the father of plant tissue culture. He
predicted that eventually a complete and functional plant could be generated from
a single cell. (Totipotency)
• Other studies led to the culture of isolated root tips.
• The approach of using explants with meristematic cells produce the successful
and indefinite culture of tomato root tips.
• Only in 1985 was the sweetening agent from the flowers and leaves of the
verbena plant, Lippia dulcis, which exceeds saccharose l000-fold in its sweetening
power and which was used already by the Aztecs to sweeten their food, identified
as a colorless sesquiterpene oil, hernandulcin.
• The psychogenically active compounds from the "holy" fungi of the Indians,
psilocybin and psilocin from the leaves of the genus Psilocybe, are now being
utilized in modern medicine due to their structural similarity to the neurotransmitter
serotonin.

14
CULTIVATION METHODS:
• Once protoplasts are separated from the isolation medium and transferred to a
suitable growth medium, adaptation and regeneration processes begin.
Adaptation is necessary because the growth conditions differ from those in the
tissue's previous environment.
Stabilization:
• The culture media for protoplasts are very similar to those for individual cells.
However, in the former it is necessary to create conditions favoring the formation
of cell walls and ensuring the stability of naked cells. Thus, the addition of
polyethylene glycol (PEG) 1500 to the medium often accelerates the uniform
deposition of micro fibrils.
Wall Formation:
• Polysaccharides such as hemicellulose synthesized for cell wall formation are
initially largely excreted into the medium. Studies on the structure of newly formed
1. PROTOPLAST CULTURE:

15
Medium Components:
Particular attention is paid to the effect of ion concentrations on protoplast division.
Depending on the origin, the auxin/cytokinin ratio must also be specifically adapted.
Highly differentiated mother cells (e.g. leaf cells) usually require a low auxin/kinetin
ratio, while protoplast cultures obtained from actively growing cell cultures or
meristematic tissue require high auxin/cytokinin ratios.
Sensitivity:
Further, a particularly great sensitivity to certain components of the medium and
environmental conditions (light, shearing forces) is characteristic of protoplast
cultures. For example, freshly isolated protoplasts are usually highly light-sensitive,
at least during the first 4-7 days.
1. PROTOPLAST CULTURE

16
The preferred donor materials for individual cells are young leaves, calli and
protoplasts. Cultures of N. tabacum and Phaseolus vulgaris, filtered off by a
mesh of 0.1 to 0.3 mm, consist of 90% single cell.
The three basic techniques:
• nurse culture
• plating technique
• culture in a microchamber
2. CULTURES OFSINGLE CELLS:

17

18
 Nurse Culture:
The isolated cells to be cultured are fixed on a piece of filter paper on the surface of
an actively growing nurse callus. The contact through the absorbent paper is
sufficient to maintain a supply of nutrients and unknown growth factors. Once the
developing microcalli have attained the size necessary for survival (200-400 µm),
they are individually cultivated on a fresh medium.

19
 Plating and Feeder-Layer Technique; Culture in a Microchamber:
• Conditioned Medium:
During a nurse culture, unknown growth factors accumulated in a suspension culture
or on the supporting filter paper are transmitted by the medium from cell to cell. In
this way, the medium is enriched with these substances. Such a medium is thus
called conditioned if it contains such substances excreted by any living cells which
ensure survival and reproduction of cells cultivated at a suboptimal density. The
effectivity of such media depends in part on the age of the nurse culture. However,
nutrients depleted from the medium during preculturing must be replaced, otherwise
they may be inhibitory.

20
• Plating and Feeder-Layer Techniques:
Plating Technique.
In this technique, cultures with a cell density less than the critical density are mixed
with the medium containing agar (0.6%) at a temperature of 30-35 °C and poured
into petri dishes to a depth of 1 mm. The low agar density makes it possible to follow
the development of single cells through an inverse microscope.
The culture may be divided into individual cubes, called agar beads. Plating success
is determined by the ratio of the number of cells in the original suspension to the
number of colonies developing within the following 21 days.
The goal is to attain the highest possible plating efficiency (PE), the number of
colonies per plated single cells, at the least possible cell density. The lower boundary
has so far been around 5000/ml.

21
• Plating and Feeder-Layer Techniques:
Feeder-Layer Technique:
In order to add a nutrient supply and stimulate division, living cells or protoplasts
unable to divide are added to the agar medium as a feeder layer.

22
• Culture in a Microchamber:
In micro chambers, the problem of insufficient cell density is solved by reducing the
amount of medium. Starting from the minimum concentration that ensures plating
success, the amount was reduced to 0.6 µl, 0.25-0.5 µl and even to microdroplets of
10-25 nl Such mini-cultures of a single cell or protoplast may be cultivated by either
hanging them from a slide held in place by the medium's surface tension (hanging
drop culture), or on a slide or in mini-agar caverns (1 x 1 x 1.75 mm) open above.

24
From the point of view of an application-oriented industry, experiments performed on
the scale of petri dishes and Erlenmeyer flasks cannot be economically utilized. Even
in laboratory fermenters with a capacity of 5-501, questions of scaling-up to industrial
scale are usually unanswerable. Therefore, industrial production conditions are
usually simulated in so-called pilot plants at a scale up to 1000 l. The results thus
obtained can be applied to larger reactors, provided the geometric and dynamic
parameters remain constant in the scaling factor. However, this is possible without
further difficulties only if a single parameter is rate-determining. This factor must be
kept constant during scaling-up.
 Determining Factors:
The aspects that must be considered in culturing large volumes may be classified as
process and culture conditions. The extent of scaling-up is determined by factors of
physical, chemical, biochemical and biological nature.
3. MASS CULTIVATION METHOD:

25
3. MASS CULTIVATION METHOD:

27
GROWTH OF CELL AND TISSUE CULTURE:
Measurement methods:
Changes in growth may be measured by fresh and dry weight, cell mass, cell
number, mitotic index or indirectly by the conductivity of the medium.
Fresh Weight: The method of fresh-weight determination neglects the various water
contents of the material. Therefore, the values of callus cultures, frequently
determined as total weight of callus, medium layer and petri dish, experience large
variations due to evaporation via the medium's surface. More exact values are
obtained by determining the weight after complete separation from the culture
medium. This is possible when the material is cultured on separating layers of
cellulose or nylon.
Dry Weight: Quantification by means of dry weight excludes error due to varying
endogenous water contents. This requires repeated drying, usally at 60°C, to the
point of constant weight. Up to fresh weights of 500 mg, a linear relationship between
fresh and dry weight is assumed.
GROWTH PROCESS:

28
Cell Mass:
This may be determined by densification by centrifugation (ca. 2000 g, 5 min) of a
particular percentage of the volume (ca. 4-7 ml) in graduated, conical centrifuge
tubes. In order to avoid errors due to water absorption by the cells, the so-called
packed cell volume (PCV) must be recorded immediately following the separation
process.
Cell Number:
To determine the number of cells per unit volume, existing cell clumps or aggregates
must be separated into isolated cells - not only in callus cultures but also in most
suspension cultures. This is commonly done using chrome-trioxide alone or in
combination with hypochlorous acid. Possible alternatives are EDT A and pectinase.
GROWTH PROCESS:

29
Conductivity:
The inverse relationship between the conductivity and fresh or dry weight of the
medium allows the determination of growth without taking samples (which would
affect the sterility of the culture). In fully synthetic media, conductivity is determined
almost exclusively by salt concentrations. As long as the pH of the medium remains
above 3 (CH + < 10-3 mol/I), the concentration of hydrogen ions does not affect
conductivity.
Cellulose Concentration:
Calcofluor-white ST (0.1 % aqueous) allows monitoring of changes in the
concentrations of cell wall polymers from fJ-glycosidic bound glucose molecules such
as cellulose or callose. The textile brightener specifically binds to fJ-l,4-glucans and
intensely fluoresces following stimulation with shortwave blue light. In this way, even
traces of these compounds may be identified
GROWTH PROCESS:

PLANT CELLS AS PRODUCERS OF
SECONDARY METABOLITES:
30

SECONDARY METABOLITES:
31
Secondary metabolites, also called specialised metabolites,
toxins, secondary products, or natural products, are organic compounds
produced by bacteria, fungi, or plants which are not directly involved in the
normal growth, development, or reproduction of the organism.
They were labelled secondary compounds in contradistinction to primary
compounds due to:
1. an apparently limited taxonomic distribution
2. Synthesis occurring only under certain conditions
3. An apparent lack of function
4. No apparent necessity for life.

32
SOME PLANT SECONDARY METABOLITES AND THEIR
APPLICATION:
Metaobolites Application Species
Ajmalicine Circulation Catharanthus roseus
Atropine Anti-cholinergic Atropa belladonna
Hyoscyamine
Hyoscine
Theophylline
Anti-cholinergic
Anti-cholinergic
Hyoscyamus spp.
Datura spp.
Camellia sinensis
Diosgenin Contraceptive Dioscorea spp.
Quinine Anti-Malarial Cinchona spp
Eugenol
Local anesthetic
Syzygium aromaticum
Morphine Analgesics Papaver somniferum

33
STORAGE OF SECONDARY METABOLITES:

34
STORAGE OF SECONDARY METABOLITES:
Accumulation:
Turnover: Depending on the object, secondary compounds are either secreted
into the surrounding medium or stored intracellularly. There they experience
turnover processes with characteristic half-lives. Their degradation was first
proven in cell suspension cultures. Degradation and synthesis often occur
simultaneously. The extent of their accumulation is mainly determined by three
cell capacities: synthetic capacity, storage capacity and the capacity to
metabolize the compounds in transport and detoxification processes.
Individual plant organs differ in their significance in this process.

35
PHARMACEUTICALLY ACTIVE NATURAL PRODUCT
SYNTHESIS VIA PLANT CELL CULTURE TECHNOLOGY:
Most secondary metabolites are often present in extremely low
amounts in the plant, often less than 1% of the total carbon.
This paucity can make natural harvestation impractical for bulk
production, especially in the case of slow growing species. The
significant engineering challenge is then to find a means by which
to produce the desired natural products in a way that is both
sustainable and financially feasible. Production of these products
in a microbial or fungal host by transferring the biosynthetic
pathway is possible.

PRODUCTION OPTIONS FOR NATURAL
PRODUCTS:
36
Chemical synthesis of natural products is possible and commercially feasible,
particularly for those with relatively simple chemical structures such as
aspirin (derived from the natural product salicylic acid) and ephedrine. In many
cases, however, the metabolite has a complex structure, which can include
multiple rings and chiral centers, so that a synthetic production process
becomes prohibitively costly. Many natural products used in cancer treatment,
including compounds such as paclitaxel, vinblastine, and camptothecin, fall into
this latter class, so an alternative method of supply is necessary.

37
PRODUCTION OPTION FOR NATURAL PRODUCTS :
• Depending on the nature of the plant, extraction directly from harvested
plant tissue may be an option. Especially if a plant can be cultivated en
masse, this can be attractive on a commercial basis.
• The anticancer drugs vincristine and vinblastine, among other medicinally
valuable metabolites such as ajmalicine and serpentine, are found in the
Madagascar periwinkle Catharanthus roseus.
• Even though these important alkaloids, particularly vincristine and
vinblastine, naturally occur at very low levels in C. roseus less than 3 g per
metric tons the fast growing nature of the periwinkle makes ﬁeld cultivation
most practical at the present time.
• However, the relative inefﬁciency and high cost of whole plant extraction
implies that an improved method of supply would be useful for these
valuable anticancer agents.

38
• When natural supply is limited due to a combination of low yields and slow
growth rates, in vitro cultures provide an attractive alternative. Most plant
species can be cultured in vitro in either an undifferentiated or differentiated
state.
• As many secondary metabolites are produced by specialized cells, organ
cultures such as shoots or roots can exhibit similar metabolite proﬁle
patterns compared to the native plant, whereas undifferentiated cultures
often accumulate secondary metabolites to a lesser extent, and sometimes
not at all.

39
• The anticancer compound camptothecin, produced by the ornamental tree
Camptotheca acuminata as well as Nothapodytes fetida and Ophiorrhiza
pumila among other species, has been shown to accumulate in
undifferentiated cultures in very low or even undetectable amounts
compared to root cultures in which production levels were comparable to the
intact plant.
• Similarly, no artemisinin, a potent antimalarial drug, was found in cell
suspension cultures of Artemisia annua, while trace amounts were detected
in shoot cultures.

40
• Root cultures can be transformed into hairy roots using the soil dwelling
bacteria Agrobacterium rhizogenes, resulting in cultures which are
genetically stable, capable of unlimited growth without additional hormones,
and have an increased capacity for secondary metabolite accumulation.
• Undifferentiated suspension cultures, which can be more easily scaled to
levels suitable for commercial production. There are currently 14 plant cell
culture processes which have been commercialized for production of
secondary metabolites (including products used in applications other than
pharmaceuticals such as food and cosmetics).

41

PLANT SUSPENSION CELL CULTURE TECHNOLOGY:
42
• Production of metabolites via plant cell suspension culture is renewable, environmentally
friendly, and from a processing standpoint, amenable to strict control, an advantage in
regards to meeting Food and Drug Administration manufacturing standards. Technology
developed for other cell culture and fermentation systems (e.g., mammalian and yeast) can
be readily adapted for large scale applications with plant cells, easing difficulties associated
with scale-up.
• A notable example of the success of plant cell culture systems, due in large part to
innovative research and the application of novel technologies, is paclitaxel synthesis and
supply. Paclitaxel, produced by Taxus spp., is an important anticancer agent used as a first
line treatment for several types of cancer, including breast, ovarian, and non small cell lung
cancer, and has also shown efficacy against AIDS-related Kaposi sacoma. Production of
paclitaxel via cell culture technology has been studied since the 1980s as an alternative
supply source to harvest of the slow growing yew tree, since a single dose of 300 mg
requires the sacrifice of a 100 year old tree

43
PLANT SUSPENSION CELL CULTURE TECHNOLOGY :

44
PLANT SUSPENSION CELL CULTURE TECHNOLOGY :
• The primary challenges impeding regular commercial application of plant
cell culture technology are low and variable yields of metabolite
accumulation.
• Some metabolites do not accumulate in appreciable quantities in
undifferentiated cells. In these cases, manipulation of genes within the
biosynthetic pathway is needed to utilize plant cell cultures for bulk
production.

45
HAIRY ROOT CULTURES ASASOURCE OF SECONDARY
METABOLITES
• The hairy root system based on inoculation with Agrobacterium
rhizogenes has become popular in the two last decades as a method of
producing secondary metabolites synthesized in plant roots.
• The hairy root phenotype is characterized by fast hormone-independent
growth, lack of geotropism, lateral branching, and genetic stability. The
secondary metabolites produced by hairy roots arising from the infection of
plant material by A. rhizogenes are the same as those usually synthesized
in intact parent roots, with similar or higher yields.
• This feature, together with genetic stability and generally rapid growth in
simple media lacking phytohormones, makes them especially suitable for
biochemical studies not easily undertaken with root cultures of an intact
plant.

46
HAIRY ROOT CULTURES ASASOURCE OF SECONDARY
METABOLITES
• During the infection process, A. rhizogenes transfers a part of the DNA
(transferred DNA, T-DNA) located in the root-inducing plasmid Ri to plant
cells, and the genes contained in this segment are expressed in the same
way as the endogenous genes of the plant cells.

47
METABOLIC ENGINEERING AND DIRECTED BIOSYNTHESIS
• The engineering of biosynthetic pathways within a plant cell
to enhance accumulation of a constitutively produced metabolite
is an appealing strategy in which exciting progress has been
made in the past decade.
• A variety of tools have been employed to both identify unknown
genes and characterize secondary metabolite pathway
regulation, including precursor feeding, gene over
expression, application of metabolic inhibitors, and mutant
selection.
• Additionally, elicitation, in relation to improving bulk yields in cell
culture, can also be used as a powerful tool to investigate
pathway regulation based on gene expression.

48
METABOLIC ENGINEERING TOOLS:
• A metabolic engineering approach involves the manipulation of
targets within a cell. Techniques are therefore needed both for
the identification of these targets (i.e., genes, proteins,
metabolites) as well as for their exploitation.
• As many secondary pathways are still partially undefined,
elucidating pathway genes and their control elements is an
active research area.
• The subsequent identification of rate influencing steps within
a biosynthetic pathway can then be useful in providing targets for
a rational engineering strategy.

49
• Plant cell cultures, including both suspension cultures and hairy
root cultures, have proven to be an extremely useful platform for
metabolic studies, as a fast growing and renewable source of
material.
• Whole plants can also be valuable, particularly as models to
study complex spatial and temporal control mechanisms
associated with environmental stimuli and morphogenesis from
a global metabolic perspective.

50
• Several approaches have been used to identify the enzymes and their
corresponding genes which catalyze biosynthetic pathway steps.
• For the paclitaxel pathway, a successful approach utilized by the Croteau
laboratory incorporated feeding cell free Taxus extracts with precursors to
isolate and identify intermediate metabolites and enzymes.
• This approach led to the identification of taxadiene synthase, which
catalyzes the first committed step of the taxane pathway.
• Genes were subsequently identified from a cDNA library using PCR
amplification based on degenerate primers designed to recognize
conserved regions from homologous enzymes in other plants whose DNA
sequences were known.

51
• Differential display methods (via reverse transcription and
PCR) comparing mRNA transcripts between elicited and
unelicited cells supplemented with a homology-based search of
a cDNA library from elicited cells, as well as random
sequencing of the same induced library, have also proven to be
extremely effective in gene discovery (for a comprehensive
review of molecular genetics in Taxus.

52
TRADITIONAL STRATEGIES TO IMPROVE CELL CULTURE
YIELDS:
Optimization of cultural conditions:
• Number of chemical and physical factors like media components,
phytohormones, pH, temperature, aeration, agitation, light affecting
production of secondary metabolites affect culture productivity.
• Several products were found to be accumulating in cultured cells at a
higher level than those in native plants through optimization of cultural
conditions. Manipulation of physical aspects and nutritional elements
in a culture is perhaps the most fundamental approach for optimization of
culture productivity.
• For example, ginsenosides by Panax ginseng and Dixon, rosmarinic acid
by Coleus bluemei, shikonin by Lithospermum, ubiquinone-10 by Nicotiana
tabacum, berberin by Coptis japonica were accumulated in much higher
levels in cultured cells than in the intact plants.

53
YIELDS:
Precursor feeding:
Exogenous supply of a biosynthetic precursor to culture medium may also
increase the yield of the desired product. This approach is useful when the
precursors are inexpensive. The concept is based on the idea that any
compound, which is an intermediate, in or at the beginning of a secondary
metabolite biosynthetic route, stands a good chance of increasing the yield of
the final product. Attempts to induce or increase the production of plant
secondary metabolites, by supplying precursor or intermediate compounds,
have been effective in many cases.

54
YIELDS:
Precursor feeding:
For example,
• amino acids have been added to cell suspension culture media for
production of tropane alkaloids, indole alkaloids etc.
• Addition of phenylalanine to Salvia officinalis cell suspension cultures
stimulated the production of rosmarinic acid.
• Addition of the same precursor resulted stimulation of taxol production in
Taxus cultures.
• Feeding ferulic acid to cultures of Vanilla planifolia resulted in increase
in vanillin accumulation.
• Furthermore, addition of leucine, led to enhancement of volatile
monoterpenes in cultures of Perilla frutiscens, where as addition of geraniol
to rose cell cultures led to accumulation of nerol and citronellol

55
YIELDS:
Elicitation:
Plants produce secondary metabolites in nature as a defense
mechanism against attack by pathogens. Elicitors are signals
triggering the formation of secondary metabolites.
Perhaps the most notable strategy for improving metabolite yields
is elicitation. An elicitor can be deﬁned as any compound that
induces an upregulation of genes. Some elicitors target secondary
metabolic genes, which are often associated with defense
responses to perceived environmental changes.

56
YIELDS:
Elicitation:
• Elicitors include natural hormones, nutrients, and many fungi-derived
compounds. In particular, jasmonic acid and its methyl ester methyl
jasmonate (MJ), are naturally occurring hormones involved in the
regulation of defense genes as part of a signal transduction system.
• Applied exogenously, they have been shown to induce secondary metabolic
activity and promote accumulation of desired metabolites in numerous plant
systems, including Taxus spp. and C. roseus.
• Different elicitors may act on different segments of the biosynthetic pathway.
For instance, MJ elicitation compared to salicylic acid elicitation in
Taxus spp. cultures resulted in different relative increases of
metabolic intermediates, suggesting that each elicitor preferentially directs
ﬂux toward, and possibly away from, different intermediate taxanes.

57
YIELDS:
Elicitation:
While many of the specific targets of elicitors have yet to be conclusively
identified, elicitation can be an extremely useful tool in conjunction with gene
expression profiling for identifying rate-influencing steps in secondary
biosynthetic pathways.

58
YIELDS:
Immobilization of plant cell cultures:
• It has long been considered for increasing metabolite accumulation, as the
potential of higher cell densities, continuous removal of products/inhibitors, and
protection for shear-sensitive plant cells provide a number of advantages.
• Immobilization can be simply achieved using a gel matrix such as alginate;
however this becomes costly at a larger scale, especially when the product of
interest is not secreted and must be released using sonication or treatments with
an organic solvent.

59
YIELDS:
Immobilization of plant cell cultures:
• Recently, immobilization of T. baccata cells in calcium-alginate beads was shown
to produce one of the highest reported levels of paclitaxel accumulation among
academic laboratories (43 mg/L).
• Immobilization also has the potential to simplify product extraction and
puriﬁcation, as immobilized cultures of Linum usitatissimum excrete the
pharmaceutically active metabolite dehydrodiconiferyl alcohol 4--D-glucoside
(DCG) to a greater extent than suspension cultures.

Stafford, A., Morris, P., & Fowler, M. (1986). Plant cell biotechnology: a perspective.
Enzyme and microbial technology, 8(10), 578-587.
Endress, R., & Endress, R. (1994). Plant cell biotechnology: Springer.
Kolewe, M. E., Gaurav, V., & Roberts, S. C. (2008). Pharmaceutically active natural
product synthesis and supply via plant cell culture technology. Molecular pharmaceutics,
5(2), 243-256.
Aboujaoude, E., Salame, W., & Naim, L. (2015). Telemental health: a status update.
World psychiatry, 14(2), 223-230.
Mulabagal, V., & Tsay, H.-S. (2004). Plant cell cultures-an alternative and efficient
source for the production of biologically important secondary metabolites. Int J Appl Sci
Eng, 2(1), 29-48.
60
REFERENCES:

THANK
YOU
Sidra
sidratulmuntaha1786@gmail.com
02332013039
M.Phil Pharmacognoy 2nd Semester

PLANT CELL BIOTECHNOLOGY: RECENT APPROACHES FOR SECONDARY METABOLITE PRODUCTION

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to PLANT CELL BIOTECHNOLOGY: RECENT APPROACHES FOR SECONDARY METABOLITE PRODUCTION

Similar to PLANT CELL BIOTECHNOLOGY: RECENT APPROACHES FOR SECONDARY METABOLITE PRODUCTION (20)

Recently uploaded

Recently uploaded (20)

PLANT CELL BIOTECHNOLOGY: RECENT APPROACHES FOR SECONDARY METABOLITE PRODUCTION