by dr ijaz ahmad

1. TISSUE CULTURE FOR
NATURAL PRODUCT
BY IJAZ AHMAD
M. PHIL IST SEMESTER

2. CONTENTS
• Introduction
• Definition of tissue culture and natural product
• History of tissue culture
• Bioreactors and types
• Elicitors and types
• Techniques used in tissue culture
• Pharmaceutical product which are obtained through tissue culture

3. TISSUE CULTURE
• Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues
or organs under sterile conditions on a nutrient culture medium of known composition.
• Plant cell/tissue culture, also referred to as in vitro, axenic, or sterile culture, is an important tool
in both basic and applied studies as well as in commercial application
• Natural product; Natural products are the products which are obtained from various natural
sources like plants, animals and microbes.
• In practice the term natural products refers to secondary metabolites produced by an organism,
but not strictly necessary for the survival of the organism. OR
• A natural product is a chemical compound or substance produced by a living organism – found
in nature that usually has a pharmacological or biological activity for use in pharmaceutical drug
discovery and drug design.

5. • METABOLITES; metabolite is an intermediate or end product of metabolism. The term metabolite is
usually used for small molecules.
PRIMARY METABOLITES: Needed for the survival, growth, development and present in all living system,
plant and animals like;
Carbohydrates
Fats
Proteins
Organic acids
Vitamins
Chlorophylls

6. SECONDARY METABOLITES:
Synthesized from primary metabolites and not needed for cell survival but
contribute to the survival of whole organism.
• Glycosides
• Phenolic compounds
• Terpenoids
• Alkaloids

7. CALLUS, CELL SUSPENSION AND PROTOPLAST:
• 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

8. EXPLANT
• 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.

9. MEDIA COMPONENTS AND PREPARATION
• INORGANIC SALTS
• The Murashige and Skoog (MS) formulation is the most widely used and will be
the major salt formulation used in these exercises.

11. PLANT GROWTH REGULATORS
• The type and concentration of plant growth regulators used will vary according to
the cell culture purpose.
• An auxin (IAA, NAA, 2,4-D, or IBA) is required by most plant cells for division and
root initiation. At high concentrations, auxin can suppress morphogenesis.
• The auxin 2,4-D is widely used for callus induction: IAA, IBA, and NAA are used for
root induction.
• Cytokinins (kinetin, BA, zeatin, and 2iP) promote cell division, shoot proliferation,
and shoot morphogenesis.

12. • Thidiazuron has been effective in low concentrations to stimulate shoot
formation.

13. VITAMINS AND CARBOHYDRATE
• Vitamins have catalytic functions in enzyme reactions.
• The vitamin considered important for plant cells is thiamine (B1).
• Other vitamins, nicotinic acid (B3) and pyridoxine (B6), are added to cell culture media,
as they may enhance cellulalular response.
• Green cells in culture are generally not photosynthetically active and require a carbon
source. Sucrose or glucose at 2–5% (w/v) is commonly used in cell culture. Other
carbohydrate sources, such as fructose and starch, can also be used.
• Lower levels of a carbohydrate may be used in protoplast culture, but much higher
levels may be used for embryo or anther culture.

14. ANTI BIOTICS AND PH
• timentin, carbenicillin (500 mg/liter), cefotaxime (300 µg/ml) and augmentin (250
mg/liter). The antibiotics are soluble in water, should be made up fresh, and
should be added to the medium after autoclaving by filter sterilization.
• The pH of plant tissue culture media is generally adjusted to pH 5.5 to 6. Below
5.5, the agar will not gel properly and above 6.0, the gel may be too firm.

15. AMINO ACIDS
• Amino acids and amines can be very important in morphogenesis.
• All l-forms of amino acids are the natural forms detected by the plant; l-tyrosine
can contribute to shoot initiation .
• l-arginine can facilitate rooting, and l-serine can be used in microspore cultures to
obtain haploid embryos.
• Amides, such as l-glutamine and l-asparagine, sometimes significantly enhance
somatic embryogenesis.

16. PLANT TISSUE CULTURE TECHNIQUES FOR
MEDICINAL PRODUCTION
Basic culture techniques
• The term “plant tissue culture” broadly refers to the in vitro and aseptic cultivation of
any plant segment, a single cell, a tissue or an organ.
• There are five main types of plant cultures including seedlings (whole plants),
embryos, organs, cells and protoplasts.
• Secondary metabolite production in plant tissue cultures have been carried out in
liquid cultures, predominantly with cell suspension cultures, mainly because liquid
cultures provide uniform culture conditions, support more rapid growth, and are more
feasible for large-scale applications.

17. • The media for plant cell cultures are usually composed of several macro-inorganic
elements (N, P, K, Mg2+ and Ca2+) and micro-elements (Fe2+, Mn2+, Zn2+,
Mo2+, Co2+, Cu2+, B3+), a few organic nutrients (vitamins and amino acids),
plant growth regulators (phytohormones), and the carbon source (sucrose or
some other carbohydrate).
• The medium composition has profound effects on the cell growth and secondary
metabolite accumulation, and needs to be optimized for specific cell lines and
production processes.
• In addition, primary metabolism and secondary metabolite biosynthesis often
require different nutrients, as many secondary metabolites are not associated with
growth.

18. • In this case, a two-stage culture process is more efficient, with the first stage
using a culture medium PLAN for maximum cell proliferation, and the second
stage for maximum secondary metabolite accumulation.
• The successful commercial production of shikonin in Lithospermum erythrorhizon
cell culture was carried out in a two-stage culture process using LS medium for
growth and M-9 medium for product formation, respectively.

19. • For experimental studies, 10–100 ml of cell suspension cultures are usually
maintained in Erlenmeyer flasks on shaking incubators, generally known as shake-
flask cultures.
• Plant cells in suspension cultures form aggregates of several to more than a few
hundred cells, with the actual aggregate size depending on the cell species,
culture environment and agitation intensity.
• A suitable degree of aggregation may be favorable for secondary metabolite
accumulation, but the formation of very large aggregates may lead to slow
growth and low productivity due to poor nutrient transfer to the centre of the
aggregates.

20. • For large-scale plant cell suspension cultures, mechanically agitated and aerated
stirred-tank reactors and air-lift reactors are the two most common types of
culture vessels.
• In a stirred-tank reactor, mechanical agitation by a rotating impeller is used to
suspend the cells, to disperse the air bubbles from an aerator, to achieve fluid
mixing, and thus good mass and heat transfer throughout the culture system.
• An air-lift reactor has no internal moving mechanical parts, but uses air sparging
as the sole means for agitation and aeration. Compared with stirred-tanks, air-lift
reactors have a simpler mechanical structure and lower energy consumption, but
are less flexible for controlling the mixing conditions and less effective for high-
density cultures.

21. MEDICINAL PLANT CELL CULTURES IN
BIOREACTORS
• A Bioreactor as a closed system used for bioprocessing (flask, roller bottle, tank,
vessel, or another container), which supports the growth of cells, mammalian or
bacterial, in a culture medium.
• a vessel which has provision of cell cultivation under sterile condition & control of
environmental conditions e.g., pH, Temperature, Dissolved oxygen etc. • It can be
used for the cultivation of microbial plant or animal cells.
• Plant cells in cultures have different morphologic and growth characteristics from
those of microorganisms, such as their relatively large size, formation of large
aggregates, sensitivity to shear stress and slow growth.

22. • bioreactor design for plant tissue cultures is to avoid product feedback inhibition
and product degradation, which can be achieved by in situ product removal from
the cells or culture medium.
• Chinese researchers have performed many studies on plant cell cultures in various
conventional bioreactors, and have also devised a number of innovative
bioreactors and culture processes.
• Pan et al. compared the culture characteristics and taxuyunnanine C yields of
Taxus chinensis cells in three bioreactors, a 1.5 L turbine-agitated reactor, a 1.5 L
bubble column reactor and a 2.5 L air-lift reactor.

23. • The cell aggregates in the turbine agitated reactor were much smaller than in the
bubble column and air-lift reactors;
• while the cell growth rates were similar in the three bioreactors, the air-lift reactor
was most favorable for taxuyunnanine production.
• High shear stress by agitation in stirred-tank reactors has been shown to cause
adverse effects on the growth and viability of Perilla frutescens cells in suspension
cultures, based on experimental results and theoretical analysis.

24. • The effects of oxygen supply on suspended cultures of P. frutescens cells were
examined in shake-flasks, a 2 L bubble column reactor and a 3 L stirred tank
reactor with a marine impeller . In all three types of culture vessels, the
production of anthocyanin pigment increased with improvement in oxygen
supply.
• it was observed that excessive aeration was harmful to the cell cultures, due
mostly to the high shear stress arising from the aeration.

25. • The immobilization of plant cells often results in a prolonged and enhanced
secondary metabolite production, and also facilitates medium perfusion (exchange)
and in situ product removal.
• Immobilized Catharanthus roseus cells in polyurethane foams have been used
continuously for a long periods of time (three weeks) for the stable production of
alkaloids.
• Immobilized Berberis julianae cells in calcium alginate gel and reticulate polyurethane
can continue the production and secretion of jatrorrhizine for about 140 days.
Lithospermum erythrorhizon cells entrapped in calcium alginate gel beads can
produce shikonin derivatives for a period of 80 days, and the total yield was higher
than that of the freely-suspended cells.

26. REACTOR TYPES AND ITS SPECIFICATION

27. ELICITATION OF SECONDARY METABOLISM
• The agents that induce plant defense responses are referred to as elicitors.
• Elicitation, the treatment of plant cells with biotic and abiotic elicitors, has been
one of the most effective means for enhancing secondary metabolite production
in plant tissue cultures.
• Biotic elicitors are usually compounds or fractions of microbial and plant cells,
and abiotic elicitors may include heavy metal ions and various forms of physical
stress such as osmotic stress, heat and cold shock, UV radiation and mechanical
stress.

28. • Among the most common and effective elicitors used for stimulating secondary
metabolite production in plant tissue cultures are the carbohydrate fractions of fungal
and plant cell walls, methyl jasmonate (MJ), chitosan and heavy metal salts.
• To find out the most effective elicitors for indole alkaloid accumulation in
Catharanthus roseus cell cultures, Zhao et al. tested the mycelial extracts of 12 fungal
species from various sources. The extracts of three Pythium species, P. irregulare, P.
aphanidermatum and P. vexans, were found to be the most effective for all three
alkaloids, ajmalicine ,serpentine ,and catharanthine ,increasing their accumulation
from 2- to 5-fold. The study also showed that the stimulating effects of various fungal
elicitors depended on the dosage and timing of elicitor treatment, and varied with
the specific alkaloid species.

29. ELICITOERS AND THEIR TYPES

30. PHARMACEUTICAL PRODUCTS
Anti-cancer drugs
• Paclitaxel (3) (Taxol) is a diterpenoid alkaloid originally isolated from the bark of
the Pacific yew, Taxus brevifolia. It is a blockbuster cancer drug for the treatment
of breast, ovarian and lung cancers, as well as AIDS-related Kaposi’s sarcoma,
because of its outstanding anticancer activity and relatively low toxicity.
• Alkaloids from Catharanthus roseus (Madagascar periwinkle) form another group
of valuable anticancer agents of plant origin, including terpenoid indole alkaloids
such as the dimeric indole alkaloids vinblastine and vincristine ,the
antihypertensive alkaloid ajmalicine ,and sedative alkaloid serpentine .

31. Cardio-vascular drugs
• Tanshinones are diterpenoid quinone pigments, and are the chief active ingredients
of Salvia miltiorrhiza roots, known as ‘danshen’ in Chinese.
• Tanshinone production has been carried out mostly in the hairy root cultures, and also
in normal and A. rhizogenes-transformed cell cultures of S. miltiorrhiza.The major
tanshinone derivatives in most of these cultures were tanshinone I ,tanshinone IIA
and cryptotanshinone .
• a callus culture of Ginkgo biloba, both ginkgolides A and B were detected, with the
ginkgolide B content varying from 0.005% to 0.01%. Ginkgolide B and bilobalide
were produced in cell cultures of G. biloba on 6,7-V medium.

32. Anti-malaria drugs –
• Artemisinin is a sesquiterpenoid isolated from the Chinese herb ‘qing hao’
(Artemisia annua). It is effective against both chloroquinine-resistant and
chloroquinine-sensitive strains of Plasmodium falciparum and against cerebral
malaria.
• At present, the commercial source of the drug is the leaves and flowering tops of
field-grown A. annua plants, which are subject to seasonal and somatic variations.
Artemisinin production has been extensively studied in shoot and hairy root
cultures.

33. Flavonoids
• Flavonoids and isoflavonoids have been produced in the tissue cultures of several
medicinal plants such as Saussurea medusa (Compositae), Maackia amurensis
(Leguminosae), Glycyrrhiza uralensis (Leguminosae), and Panax japonicus var.
major (Araliaceae). In S. medusa cell cultures, the levels of two flavonoids,
hispidulin and jaceosidin ,in the cultured cells were found to be 1.46% and 0.01%,
respectively.

34. Antioxidants
• Water-soluble phenolic acids in the Salvia plants such as lithospermic acid B (LAB, 33)
and rosmarinic acid (RA, 34) are potent antioxidants exhibiting several
pharmacological activities such as uremia- and hepatitis-preventive effects, and
endothelium dependent vasodilatory and hypotensive effects.
• LAB and RA have been produced in hairy root cultures, as well as A. rhizogenes
transformed cell cultures of S. miltiorrhiza., Lycopene is a carotenoid with strong
antioxidant activity, and has been produced in a cell culture of carrots (Daucus carota)
in a medium supplemented with 1-naphthaleneacetic acid (NAA). Vitamin E (a-
tocopherol, 28), the most well-known antioxidant, has been produced in safflower
(Carthamus tinctorius) cell cultures.

40. Morphine and Codeine
• Latex from the opium poppy, Papaver somniferum, is a commercial source of the
analgesics, morphine and codeine. Callus and suspension cultures of P. somniferum
are being investigated as an alternative means for production of these compounds.
Production of morphine and codeine in morphologically undifferentiated cultures has
been reported .Removal of exogenous hormones from large-scale culture systems
could be implemented using a two-stage process strategy .Without exogenous
hormones, maximum codeine and morphine concentrations were 3.0 mg/g dry weight
and 2.5 mg/g dry weight, respectively, up to three times higher than in cultures
supplied with hormones.

41. Camptothecin
• Camptothecin, a potent antitumor alkaloid was isolated from Camptotheca
acuminata. induced C. acuminata callus on MS medium containing 0.2 mg/l 2,4-D
and 1 mg/l kinetin and developed liquid cultures in the presence of gibberellin, L-
tryptophan, and conditioned medium, which yielded camptothecin at about
0.0025% on a dry weight basis.
• When the cultures were grown on MS medium containing 4 mg/l NAA,
accumulation of camptothecin reached 0.998 mg/l .