Plant tissue culture involves growing plant cells, tissues or organs in sterile conditions on a nutrient culture medium of inorganic salts and vitamins. The key steps are surface sterilizing explants, culturing them on growth media supplemented with hormones and sugars, and subculturing the resulting callus or cells regularly. Common explants used are roots, leaves, meristems and anthers. The process requires sterile techniques and controlled conditions to prevent contamination and maintain optimal growth.

1. PLANT TISSUE CULTURE
SATYAJIT GHOSH
B. PHARM 4TH
SEMESTER

2.  Introduction: -
Tissue culture is in vitro cultivation of plant cell or tissue under aseptic and controlled environmental
conditions, in liquid or on semisolid well-defined nutrient medium for the production of primary and
secondary metabolites or to regenerate plant.
The whole process requires a well-equipped culture laboratory and nutrient medium. This process
involves various steps, viz. preparation of nutrient medium containing inorganic and organic salts,
supplemented with vitamins, plant growth hormone(s) and amino acids as well as sterilization of explant
(source of plant tissue), glassware and other accessories inoculation and incubation.
 General procedures involved in plant tissue culture: -
In vitro culturing of plant tissue involves the following steps:
a- Sterilization of glassware tools/vessels
b- Preparation and sterilization of explant
c- Production of callus from explant
d- Proliferation of cultured callus
e- Sub-culturing of callus
f- Suspension culture
 Sterilization of Glassware Tools/Vessels: -
i. All the glassware to be used in tissue culture laboratory should be of pyrex or corning. To make them
free from any dirt, waxy material or bacteria, all the glassware should be kept overnight dipped in
sodium dichromate-sulphuric acid solution.
ii. Next morning, glassware should be washed with fresh running tap water, followed by distilled water
and placed in inverted position in plastic bucket or trays to remove the extra water.
iii. For drying the glassware, it is placed in hot air oven at high temperature about 120°C for 0.5–1 h.
iv. In the case of plastic labware, washing should be carried out with a mild nonabrasive detergent
followed by washing under tap water or the plasticware after general washing with dilute sodium
bicarbonate and water followed by drainage of extra water, rinsed with an organic solvent such as
alcohol, acetone and chloroform.
v. To prevent reinfection following sterilization, empty containers are wrapped with aluminum foil.
Stainless steel, metal tools (knives, scalpels, forceps, etc.) are also wrapped with the aluminum foil

3. and pads of cotton wool are stuffed into the opening of the pipettes, which are either also wrapped in
aluminum or placed in an aluminum or stainless-steel box.
 Preparation of Explant: -
i. Explant can be defined as a portion of plant body, which has been taken from the plant to establish a
culture. Explant can be obtained from plants, which are grown in controlled environmental
conditions. Such plants will be usually free from pathogens and are homozygous in nature.
ii. Explant may be taken from any part of the plant like root, stem, leaf, or meristematic tissue like
cambium, floral parts like anthers, stamens, etc.
iii. Age of the explant is also an important factor in callus production. Young tissues are more suitable
than mature tissues. A suitable portion from the plant is removed with the help of sharp knife, and
the dried and mature portions are separated from young tissue.
iv. When seeds and grains are used for explant preparation, they are directly sterilized and put in
nutrient medium. After germination, the obtained seedlings are to be used for explant preparation.
 Surface Sterilization of Explant: -
i. For the surface sterilization of the explant, chromic acid, mercuric chloride (0.11%), calcium
hypochlorite, sodium hypochlorite (1–2%) and alcohol (70%) are used.
ii. Usually the tissue is immersed in the solution of sterilizing agent for 10 to 15 min, and then they are
washed with distilled water. Repeat the treatment with sodium hypochlorite for 20 min, and the
tissue is finally washed with sterile water to remove sodium hypochlorite. Such tissue is used for
inoculation.
iii. In the case of leaf or green fresh stem the explant needs pretreatment with wetting agent (70–90%
ethyl alcohol, Tween 20), 5–20 drops in 100 ml of purified water or some other mild detergent to be
added directly into the sterilization solution to reduce the water repulsion.
 Production of Callus from Explant: -
i. The sterilized explant is transferred aseptically onto defined medium contained in flasks. The flasks
are transferred to BOD incubator for maintenance of culture.
ii. Temperature is adjusted to 25±2℃. Some amount of light is necessary for callus production. Usually
sufficient amount of callus is produced within three to eight days of incubation.

4.  Proliferation of Callus: -
i. If callus is well developed, it should be cut into small pieces and transferred to another fresh medium
containing an altered composition of hormones, which supports growth. The medium used for
production of more amount of callus is called proliferation medium.
 Sub-culturing of Callus: -
i. After sufficient growth of callus, it should be periodically transferred to fresh medium to maintain
the viability of cells. This sub-culturing will be done at an interval of 4–6 weeks.
 Suspension Culture: -
i. Suspension culture contains a uniform suspension of separate cells in liquid medium. For the
preparation of suspension culture, callus is transferred to liquid medium, which is agitated
continuously to keep the cells separate.
ii. Agitation can be achieved by rotary shaker system attached within the incubator at a rate of 50–150
rpm. After the production of sufficient number of cells sub-culturing can be done.
 Culture media: -
 Media composition: -
To maintain the vital functions of a culture, the basic medium consisting of inorganic nutrients
(macronutrients and micronutrients), organic components (amino acids, vitamins), growth regulators
(phytohormones) and utilizable carbon (sugar) source and a gelling agent (agar/phytogel).
A- Inorganic nutrients: -
Mineral elements play very important role in the growth of a plant. For example, magnesium is a
part of chlorophyll molecule, calcium is a component of cell wall and nitrogen is an important
element of amino acids, vitamins, proteins and nucleic acids.
Iron, zinc and molybdenum are parts of certain enzymes. Essentially about 15 elements found
important for whole plant growth have also been proved necessary for the growth of tissue in culture.
a- Macronutrients:
The macronutrients include six major elements: nitrogen (N), phosphorus (P), potassium (K),
calcium (Ca), magnesium (Mg) and sulphur (S) present as salts in media. The concentration of
the major elements like calcium, phosphorus, sulphur and magnesium should be in the range of

5. 1–3 mmol / L whereas the nitrogen in the media (contributed by both nitrate and ammonia)
should be 2–20 mmol / L.
b- Micronutrients:
The inorganic elements required in small quantities but essential for proper growth of plant cells
or tissues are boron (B), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn) and molybdenum
(Mo).
Out of these, iron seems more critical as it is used in chelated forms of iron and zinc in preparing
the culture media, as iron tartrate and citrate are difficult to dissolve. The concentration
generally prescribed for all these elements are in traces.
These are added to culture media depending upon the requirement of the objective. In addition
to these elements, certain media are also enriched with cobalt (Co), iodine (I) and sodium (Na)
but exact cell growth requirement is not well established.
B- Organic nutrients: -
a- Nitrogenous substances:
Most cultured plant cells are capable of synthesizing essential vitamins but not in sufficient
amount. To achieve best growth, it is essential to suppl the tissue culture medium with one or
more vitamins and amino acid.
Among the essential vitamins’ thiamine (vitamin B1) has been proved to be essential ingredient.
Other vitamins, especially pyridoxine (vitamin B6), nicotinic acid (vitamin B3) and calcium
pantothenate (vitamin B5) and inositol are also known to improve growth of the tissue culture
material.
b- Carbon source:
It is essential to supplement the tissue culture media with a utilizable source of carbon to the
culture media. The most commonly used carbon source is sucrose at a concentration of 2–5%.
Autoclaved sucrose was better than filtered sterilized sucrose. Autoclaving may do the hydrolysis
of the sucrose thereby converting it into more efficiently utilizable sugar such as fructose.
In general, excised dicotyledonous roots grow better with sucrose whereas monocots do best
with dextrose (glucose). Glucose and fructose are also known to be used for good growth of some
tissues.

6. Some other forms of carbon that plant tissues are known to utilize include maltose, galactose,
mannose, lactose and sorbitol.
C- Plant growth regulators: -
The plant growth regulators used most commonly are plant hormones or their synthetic analogues.
There are five main classes of plant growth regulator used in plant cell culture, namely: (1) auxins,
(2) cytokinins, (3) gibberellins, (4) abscisic acid and (5) ethylene.
Hormones Notes
Auxins Auxins promote both cell division and cell growth. The most important naturally
occurring auxin is IAA (indole-3-acetic acid), but its use in plant cell culture
media is limited because it is unstable to both heat and light.
Occasionally, amino acid conjugates of IAA (such as indole– acetyl–L-alanine and
indole–acetyl–L-glycine), which are more stable, are used.
2,4-Dichlorophenoxyacetic acid (2,4-D) is the most commonly used auxin, and is
extremely effective in most circumstances.
Cytokinins Cytokinins promote cell division. Of the naturally occurring cytokinins, two have
use in plant tissue culture media, are zeatin and 2iP (2-isopentyl adenine).
Their use is not widespread as they are expensive (particularly zeatin) and
relatively unstable. The synthetic analogues, kinetin and BAP (benzyl
aminopurine), are therefore used more frequently.
Gibberellins They are involved in regulating cell elongation, and are agronomically important
in determining plant height and fruit set. Only a few of the gibberellins are used in
plant tissue culture media, GA3 being the most common.
Abscisic acid Abscisic acid (ABA) inhibits cell division. It is most commonly used in plant
tissue culture to promote distinct developmental pathways such as somatic
embryogenesis.
Ethylene Ethylene is a gaseous, naturally occurring, plant growth regulator most commonly
associated with controlling fruit ripening in climacteric fruits, and its use in plant
tissue culture is not widespread.
Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can
inhibit the growth and development of the culture.

7. D- Solidifying agents for solidification of the media: -
Due to improved oxygen supply and support to the culture growth, solid media are often preferred to
liquid cultures. For this purpose, substance with strong gelling capacity is added into the liquid
media. These reversibly bind water and thus ensure the humidity of the medium desired for
culturing depending on the concentration.
The most commonly used substance for this purpose is the phycocolloid agar–agar obtained from red
algae (Gelidium, Gracilaria). It is generally used at a concentration of 0.8–1.0%, with higher
concentration medium becoming hard and does not allow the diffusion of nutrients into the tissues
medium.
Agar (Agarose) is extraordinary resistant to enzymatic hydrolysis at incubation temperature, and
only a few bacteria exist which are capable of producing degrading enzyme— agarase. This
resistance to hydrolysis is the fundamental importance to the use of agar–agar in cell culture
medium.
pH of the medium is generally adjusted between 5.0 and 6.0 before sterilization. In general pH
higher than 6.0 gives fairly hard medium and pH below 5.0 does not allow satisfactory gelling of the
Agar.
 Media preparation: -
For media preparation, there are two possible methods, i.e.:
a- To weigh the required quantity of nutrient, dissolve them separately and mix at the time of medium
preparation.
b- To prepare the stock solution separately for macro- nutrients, micro-nutrients, iron solution and
organic components are stored in the refrigerator till not used.

8.  Procedure:
All the ingredients may be grouped into following four groups:
Stock solution
ingredients
Amounts
(mg / L)
Stock solution
ingredients
Amounts
(mg / L)
Group – I Group – III
NH4NO3 1650 FeSO4. 7H2O 27.8
KNO3 1900 Na2 EDTA. 2H2O 37.3
CaCl2. 2H2O 440 Group – IV
MgSO4. 7H2O 370 Inositol 100
KH2PO4 170 Nicotinic acid 0.5
Group – II Pyridoxine HCl 0.5
KI 0.83 Thiamine HCl 0.1
H3BO3 6.2 Glycine 2
MnSO4. 4H2O 22.3
ZnSO4. 7H2O 8.6
Na2MoO4. 2H2O 0.25
CuSO4. 5H2O 0.025
CoCl2. 6H2O 0.025
A- Concentration of the ingredients: -
For the preparation of stock solution, the Group I ingredient are prepared at 20x concentrated
solution, Group II at 200x, Group III Iron salts at 200x and Group IV organic ingredient except
sucrose at 200x.
B- Solution preparation: -
For the preparation of stock solution, each component should be weighed and dissolved separately
in glass distilled or demineralized water and then mixed together.
Stock solution may be prepared at the strength of 1 mmol / L or 10 mmol / L. All the stock solutions
are stored in refrigerator till used. For iron solution, dissolve FeSO4. 7H2O and Na2EDTA. 2H2O
separately in about 450 ml distilled water by heating and constant stirring. Mix the two solutions,
adjust pH of the medium to 5.5 and final volume adjusted to 1 L with distilled water.

9. C- Semisolid media preparation: -
Required quantities of agar and sucrose are weighed and dissolved in wafer by 3/4th
volume of
medium, by heating them on water bath. Adequate quantities of stock solution and other special
supplements are added and final volume is made up with double distilled water. After mixing well,
pH of the medium is adjusted to 5.8 using 0.1 N NaOH and 0.1 N HCl.
 Sterilization of Culture Media: -
Culture media packed in glass containers or vessels are sealed with cotton plugs and covered with
aluminum foils and are autoclaved at pressure of 2 – 2.2 atm at 121°C for 15–40 min.
Minimum autoclaving time includes the time required for the liquid volume to reach the sterilizing
temperature (121°C) and 15 min at this temperature.
The actual success of sterilization can be tested using a bio-indicator, commonly spores of the bacterium
Bacillus stearothermophilus are used as such as a test organism.
Together with culture medium and a pH indicator in ampoules sealed by melting, both autoclaved
material and non-autoclaved controls are incubated for 24–48 h at 60°C. If the spores are dead, the
colour of the pH indicator in the solution remains unchanged indicating no change in pH.
 Types of culture: -
Culture type Note
Root Tip Culture
Tips of the lateral roots are sterilized, excised and transferred to fresh medium.
The lateral roots continue to grow and provide several roots, which after seven
days, are used to initiate stock or experimental cultures. Thus, the root
material derived from a single radicle could be multiplied and maintained in
continuous culture; such genetically uniform root cultures are referred to as a
clone of isolated roots.
Leaves or Leaf
Primordia Culture
Leaves (800 μm) may be detached from shoots, surface sterilized and placed
on a solidified medium where they will remain in a healthy condition for a long
period.
Growth rate in culture depends on their stage of maturity at excision. Young
leaves have more growth potential than the nearly mature ones.

10. Shoot Tip Culture
The excised shoot tips (100–1000 μm long) of many plant species can be
cultured on relatively simple nutrient media containing growth hormones and
will often form roots and develop into whole plants
Anther and Pollens
Culture
Young flower buds are removed from the plant and surface sterilized. The
anthers are then carefully excised and transferred to an appropriate nutrient
medium.
Immature stage usually grows abnormally and there is no development of
pollen grains from pollen mother cells.
Anther at a very young stage and late stage of development are generally
ineffective, and hence, for better response always select mature anther or
pollen.
Mature anther or pollen grains (microspore) of several species of
gymnosperms can be induced to form callus by spreading them out on the
surface of a suitable agar media.
Mature pollen grains of angiosperms do not usually form callus, although there
are one or two exceptions.
Ovule and Embryo
Culture
Embryo is dissected from the ovule and put into culture media. Very small
globular embryos require a delicate balance of the hormones.
Hence, mature embryos are excised from ripened seeds and cultured mainly to
avoid inhibition in the seed for germination.
This type of culture is relatively easy as the embryos require a simple nutrient
medium containing mineral salts, sugar and agar for growth and development.
The seeds are treated with 70% alcohol for about 2 min, washed with sterile
distilled water, treated with surface sterilizing agent for specific period, once
again rinsed with sterilized distilled water and kept for germination by placing
them on double layers of pre-sterilized filter paper placed in petri dish
moistened with sterilized distilled water or placed on moistened cotton swab
in petri dish. The seeds are germinated in dark at 25–28°C and small part of
the seedling is utilized for the initiation of callus.

11. Hairy Root Culture
A large number of small fine hairy roots covered with root, hairs originate
directly from the explant in response to Agrobacterium rhizogenes infection
are termed hairy roots.
These are fast-growing, highly branched adventitious roots at the site of
infection and can grow even on a hormone-free culture medium.
Many plant cell culture systems, which did not produce adequate amount of
desired compounds, are being reinvestigated using hairy root culture methods.
One of the most important characteristics of the transformed roots is their
capability to synthesize secondary metabolites specific to that plant species
from which they have been developed.
Growth kinetics and secondary metabolite production by hairy roots is highly
stable and are of equal level and even they are higher to those of field grown
plants.
 Establishment and maintenance of various cultures: -
For the growth establishment and maintenance of various types of plant tissue cultures, there are two
main culture systems, selected on the basis of the objective.
a- Callus culture also known as static culture
b- Suspension culture
 Callus culture:
Callus is an amorphous aggregate of loosely arranged parenchyma cells, which proliferate from mother
cells. Cultivation of callus usually on a solidified nutrient medium under aseptic conditions is known as
callus culture.
A- Initiation of callus culture: -
a- Selection and preparation of explant: -
Selection: For the preparation of callus culture, organ or culture is selected such as segments of
root or stem, leaf primordia, flower structure or fruit, etc.
Preparation:
1. Excised parts of the plant organ are first washed with tap water, and then sterilized with
0.1% of mercuric chloride (HgCl2) or 2% w/v, sodium hypochlorite (NaOCl) solution for 15

12. min. In the case of plant organ containing waxy layer, the material is either pretreated with
wetting agents (ethanol 70–90%, tween 20); or other detergents are added to the
sterilization solution to reduce the water repulsion.
2. Wash the sterilized explants with sterile glass distilled water and cut aseptically into small
segments (2–5 mm).
b- Selection of culture medium: -
1. The culture medium depends on the species of the plant and the objective of the experiment.
The MS medium is quite suitable for dicot tissues because of relatively high concentration of
nitrate, potassium and ammonium ions in comparison to other media. Growth hormones
(auxin, cytokinin) are adjusted in the medium according to the objective of the culture. For
example, auxins, IBA and NAA are widely used in medium for rooting and in combination
with cytokinin for shoot proliferation. 2, 4-D and 2, 4, 5-T are effective for good growth of
the callus culture.
2. This is also quite favourable for monocot tissues or explant. The selected semisolid nutrient
is prepared. The pH of the medium is adjusted (5.0–6.0) and poured into culture vessels,
plugged and sterilized by autoclaving.
c- Transfer of explant: -
Surface sterilized organs (explant) from stem, root or tuber or leaf, etc., are transferred
aseptically into the vessel containing semisolid culture medium.
d- Incubation of culture: -
The inoculated vessels are transferred into BOD incubator with auto controlled device. Incubate
at 25–28°C using light and dark cycles for 12-h duration.
Nutrient medium is supplemented with auxin to induce cell division. After three to four weeks,
callus should be about five times the size of the explant.
B- Induction: -
During this stage, metabolic activities of the cell will increase; with the result, the cell accumulates
organic contents and finally divides into a number of cells.
The length of this phase depends upon the functional potential of the explant and the environmental
conditions of the cell division stage.

13. C- Cell division: -
This is the phase of active cell division as the explant cells revert to meristematic state.
D- Cell differentiation: -
This is the phase of cellular differentiation, i.e. morphological and physiological differentiation occur
leading to the formation of secondary metabolites.
E- Maintenance: -
After sufficient time of callus growth on the same medium following change will occur
i. Depletion of nutrients in the medium
ii. Gradual loss of water
iii. Accumulation of metabolic toxins
Hence for the maintenance of growth in callus culture, it becomes necessary to sub-culture the callus
into a fresh medium. Healthy callus tissue of sufficient size (5–10 mm in diameter) and weight (20–
100 mg) is transferred under aseptic conditions to fresh medium.
Sub-culturing should be repeated after even four to five weeks. Many callus cultures however remain
healthy and continue to grow at slow rate for much longer period without sub-culturing, if the
incubation is to be carried out at low temperature, 5–10°C below the normal temperature (16–18°C).
Normally, total depletion takes about 28 days.
 Suspension Culture: -
Suspension culture contains a uniform suspension of separate cells in liquid medium. For the
preparation of suspension culture, callus fragments are transferred to liquid medium (without agar),
which is agitated continuously to keep the cells separate.
Agitation can be achieved by rotary shaker system attached within the BOD incubator at a rate of 50–
150 rpm. After sufficient numbers of cells are produced, sub-culturing can be done in fresh liquid
medium. Single cells can also be obtained from fresh plant organ (leaf).
A- Initiation of suspension culture: -
a- Isolation of single cell from callus culture:
Healthy callus tissue is selected and placed in a petridish on a sterile filter paper and cut into
small pieces with the help of sterile scalpel. Selected small piece of callus fragment about 300–
500 mg and transferred into flask containing about 60 ml of liquid nutrient media (i.e. defined

14. nutrient medium without gelling agent), the flask is agitated at 50–150 rpm to make the
separation of the cells in the medium.
Decant the medium and resuspend residue by gently rotating the flask, and finally transfer 1/4th
of the entire residue to fresh medium, followed by sieving the medium to obtain the degree of
uniformity of cells.
b- Isolation of single cell from plant organ:
From the plant organ (leaf tissue) single cell can be isolated by any of the following methods:
Mechanical method:
The surface sterilized fresh leaves are grinded in (1:4) grinding medium (20 μmol sucrose; 10
μmol MgCl2, 20 μmol tris-HCl buffer, pH 7.8) in glass pestle mortar.
The homogenate is passed through muslins cloth, washed with sterile distilled water, centrifuged
with culture medium, sieved and placed on culture dish for inoculation.
Enzymatic method:
Leaves are taken from 60- to 80-day-old plant and sterilized by immersing them in 70% ethanol
solution followed by hypochlorite solution treatment, washed with sterile double distilled water,
placed on sterile tile and peeled off the lower surface with sterile forceps. Cut the peeled surface
area of the leaves into small pieces (4 cm2
). Transfer them into an Erlenmeyer flask (100 ml)
containing about 20 ml of filtered sterilized enzyme solution (macerozyme 0.5% solution, 0.8%
mannitol and 1% potassium dextran sulphate).
Incubate the flask at 25°C for 2 h. During incubation, change the enzyme solution with the fresh
one at every 30 min, wash the cell twice with culture medium and place them in culture dish.
B- Growth pattern of suspension culture: -
Growth patterns of the cells have following phases:

15. a- Lag Phase:
In this phase, after sub-culturing in a fresh medium, the cell regains division ability and the
growth of tissues slow down.
b- Exponential Phase:
In this phase, rapid cell division occurs. Its duration depends on the cell and its nutrient regime.
Mostly the duration of this phase is short, lasting only for 3-4 generations.
c- Linear Phase:
In this phase, the growth occurs in a pattern linear to time.
d- Progressive Deceleration Phase:
In this phase, the cell division rate delays with the ageing of culture.
e- Stationary Phase:
In this phase, the production rate of cells and their death rate becomes equal.
f- Senescent Phase:
In this phase, the cells start dying.
C- Maintenance of suspension culture: -
Maintenance of suspension culture can be done by following three ways:
a- Batch suspension culture:
In this technique, the cells are allowed to multiply in liquid medium, which is continuously
agitated to break up cell aggregates. In this technique to commence the growth again on the
Exponential
Linear
Progressive deceleration
Lag
Phase

16. stationary phase, more amount of nutrient medium is added to the original culture or the cells
are to be transferred into fresh medium.
Each fresh medium containing culture (suspension) constitutes a batch. Such cultures are grown
again and again for the purpose of experiment to achieve certain specific objectives.
In batch culture there is no steady state of growth; hence, it is not ideal for commercial
production of secondary metabolites.
b- Semi continuous suspension culture:
In this type, the system is open. It is designed for periodic removal of culture and addition of
fresh medium. Hence, the growth is continuously maintained.
c- Continuous suspension culture:
Here, the volume of culture remains constant and fresh medium and culture are continuously
added and withdrawn respectively. The important feature of the continuous culture is the
proliferation of cell occurs under constant conditions.
In this very suspension culture technique, a steady state is achieved by adding medium in which
single nutrient has been adjusted so as to be growth limiting. Continuous culture is closed and
open type.
In the closed type, addition of fresh medium is balanced by the outflow of spent medium. The
cell passing through the outgoing medium are separated mechanically and reintroduced into the
culture for the continuous growth of the cell biomass.
Open continuous system involves regulated new medium and balancing harvest of equal volume
of culture. The open system is further of two types depending upon regulation technique:
chemostat and turbidostat.

17.  Applications of plant tissue culture: -
 Biochemical Conversion (Bio-transformation): -
The conversion of small part of a chemical molecule by means of biological systems is termed bio-
transformation. It is a process in which the substrate can be modified.
For example, Digitalis lanata cell cultures have ability to effect hydroxylation, acetylation, glycosylation,
etc. It is reported that D. lanata strain 291 can convert β-methyl digitoxin into β-methyl digoxin.
Cell suspension culture of Strophanthus gratus affects various biochemical conversions of digitoxigenin.
Monoterpene bio-conversions are reported with mentha cell culture. It can convert (-) menthone to (+)
neomenthol and pulegone to iso-menthone.
Podophyllum peltatum in semi-continuous culture can produce anticancer drugs by bio-transformation of
synthetic dibenzyl butanolides to lignans—suitable for conversion to etoposide.
 Clonal Propagation (Micro-Propagation): -
Clonal propagation (micro-propagation) is the technique to produce entire plant from single individual
by asexual reproduction. This fact can be commercially utilized to produce high-yielding crops of the
desirable characters in a short period of time, which otherwise show variation when grown using seeds.
For example, Foeniculum vulgare (fennel) shows wide variations in the yield and composition of the
volatile oil, and by this technique, it has been reported to have uniform clones of fennel with narrow
variation in the volatile oil composition, in comparison to the normal cultivation.
 Immobilization of Plant Cells: -
The immobilization of plant cell or enzymes has increased the utility of plant cell biotechnology for
production of pharmaceuticals. The plant cells can be immobilized by using matrices, such as alginates,
polyacrylamides, agarose and polyurethane fibres.
The immobilized plant cells can be utilized in the same way as immobilized enzymes to effect different
reactions. Immobilized cell systems may be used for bio-conversions, such as (-) codeinone to (-)
codeinine and digitoxin to digoxin or for synthesis from added precursors, e.g. production of ajmalicine
from tryptamine and secologanin.
The suspension cultures of Anisodus tanguticus have been reported to convert hyoscyamine to
anisodamine in good quantity.