Hello ever one i hope its useful for preparation of notes regarding plant tissue culture for Pharmacognosy .. B.pharm II yr IV sem.. plz give comments it may useful for me and i can rectify the things.

1. PLANT TISSUE CULTURE
S.SELVADURAI, M.Pharm.,MBA.,
Asst.Professor
Dept of Pharmacognosy

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.
This technique affords alternative
solution to problems arising due to
current rate of extinction and
decimation of flora and ecosystem.

3. Advantages of Tissue Culture Technique over
the Conventional Cultivation Techniques.
•Availability of raw material
•Fluctuation in supplies and quality
•Patent rights
•Political reasons
•Easy purification of the compound
•Modifications in chemical structure
•Disease-free and desired propagule
•Crop improvement
•Biosynthetic pathway
•Immobilization of cells

4. BASIC REQUIREMENTS FOR A
TISSUE CULTURE LABORATORY
 Equipment and apparatus
 Washing and storage facilities
 Media preparation room
 Sterilization room
 Aseptic chamber for culture
 Culture rooms or incubators fully equipped with
temperature,
 light and humidity control devices.
 Observation or recording area well equipped with
computer for data processing.

5. EQUIPMENT AND APPARATUS
 Culture vessels and glassware
Many different kinds of vessels may be used
for wing cultures.
Callus culture can be grown successfully in
large test tubes (25 × 150 mm) or wide mouth
conical flasks (Erlenmeyer flask).
In addition to the culture vessels, glassware
such as graduated pipettes, measuring
cylinders, beakers, filters, funnel and petri
dishes are also required for making
preparations.

6. EQUIPMENT
 A spirit burner or gas micro-burner for flame
sterilization of instruments
 An autoclave to sterilize the media.
 Hot air oven for the sterilization of glassware, etc.
 A pH meter for adjusting the pH of the medium.
 A shaker to maintain cell suspension culture.
 A balance to weigh various nutrients for the
preparation of the medium.
 Incubating chamber or laminar airflow with UV light
fitting for aseptic transfer of explants to the medium
and for sub-culturing.
 A BOD incubator for maintaining constant temperature
to facilitate the culture of callus and its subsequent
maintenance.

7. WASHING AND STORAGE
FACILITIES
First and foremost requirement of the
tissue culture laboratory is provision for
fresh water supply and disposal of the
waste water, and space for distillation
unit for the supply of distilled and double
distilled water and de-ionized water.
Acid and alkali resistant sink or wash
basin for apparatus / equipment washing
and the working table should also be acid-
and alkali-resistant.

8. MEDIA PREPARATION ROOM
Media preparation room should have
sufficient space to accommodate
chemicals, lab ware, culture vessels and
equipments required for weighing and
mixing, hot plate, pH meter, water baths,
Bunsen burners with gas supply,
microwave oven, autoclave or domestic
pressure cooker, refrigerator and freezer
for storage of prepared media and stock
solutions.

9. STERILIZATION ROOM
For the sterilization of culture
media, a good quality ISI mark
autoclave is required and for small
amount domestic pressure cookers,
can also serve the purpose. For the
sterilization of glassware and
metallic equipments hot air oven
with adjustable tray is required.

10. ASEPTIC CHAMBER/AREA FOR TRANSFER
OF CULTURE
 For the transfer of culture into sterilized media,
contaminant - free environment is mandatory.
 modern laboratory have laminar airflow cabinet having
vertical or horizontal airflow, arrange over the working
surface to make it free from dust particles / micro-
contaminants.
 Inside the cabinet, there is arrangement for Bunsen
burner and a UV tube fitted on the ceiling of the
cabinet.
 The advantage of working in the laminar airflow cabinet
is that the flow of air does not hamper the use of Bunsen
burner and moreover, the cabinet occupies relatively
small space within the laboratory.

11. INCUBATION ROOM OR
INCUBATOR
 BOD incubators required to maintain the culture
conditions should have the following characteristics:
Temperature range, 2–40°C
Temperature
Automatic digital temperature recorder
24-h temperature and light programming
Adjustable fluorescent lighting up to 10,000
lux
Relative humidity range 20–98%
Relative humidity control ―3%

12. DATA COLLECTION AND RECORDING THE
OBSERVATION
The growth and maintenance of the
tissue culture in the incubator should be
observed and recorded at regular
intervals.
All the observations should be done in
aseptic environment, i.e. in the laminar
airflow. Whereas for microscopic
examination, separate dust-free space
should be marked for microscopic work.
All the recorded data should be fed into
the computer.

13. GENERAL PROCEDURES INVOLVED IN
PLANT TISSUE CULTURE
 In vitro culturing of plant tissue involves the following
steps:
 Sterilization of glassware tools/vessels
 Preparation and sterilization of explant
 Production of callus from explant
 Proliferation of cultured callus
 Sub-culturing of callus
 Suspension culture

14. TYPES OF PLANT TISSUE CULTURES
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.

15. Leaves or Leaf Primordia Culture
Leaves (800 μm) may be detached
from shoots, surface sterilized and
placed on a solidified medium where
they will remains in a healthy
conditions for a long periods.
Growth rate in culture depends on
their stage of maturity at excision.
Young leaves have more growth
potential than the nearly mature ones.

16. 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.

18. Complete Flower Culture
Nitsch in 1951 reported the successful culture of
the flowers of several dicotyledonous species; the
flowers remain healthy and develop normally to
produce mature fruits.
Flowers (2 days after pollination) are excised,
sterilized by immersion in 5% calcium
hypochlorite, washed with sterilized water and
transferred to culture tubes containing an agar
medium.
Often fruits that develop are smaller than their
natural counterpart, but the size can be increased
by supplementing the medium with an
appropriate combination of growth hormones.

19.  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 (containing microspore
mother cells or tetrads) and late stage (containing
binucleate starch-filled pollen) of development are
generally ineffective, and hence, for better response always
select mature anther or pollen.
 Mature anther or pollen grains (microspora) of several
species of gymnosperms can be induced to form callus b
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.

20. 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
presterilized filter paper placed in petridish moistened with
sterilized distilled water or placed on moistened cotton swab in
petridish.
 The seeds are germinated in dark at 25–28°C and small part of the
seedling is utilized for the initiation of callus.
 Apart from above-mentioned cultures, there are two more methods
for culturing of plant tissues/cells:
 Protoplast culture and
 Hairy roots culture.

21. PROTOPLAST CULTURE
 Protoplasts are the naked cells of varied origin without
cell walls, which are cultivated in liquid as well as solid
media.
 Protoplasts can be isolated by mechanical or enzymatic
method from almost all parts of the plant: roots, tubers,
root nodules, leaves, fruits, endosperms, crown gall
tissues, pollen mother cells and the cells of the callus
tissue but the most appropriate is the leaves of the plant.
 Fully expanded young leaves from the healthy plant are
collected, washed with running tap water and sterilized
by dipping in 70% ethanol for about a minute and then
treated with 2% solution of sodium hypochlorite for 20–
30 min, and washed with sterile distilled water to make
it free from the trace of sodium hypochlorite.

22. Leaf sterilization
Epidermis peeling
Peeled piece of leaf
Cell in enzyme mixture
Partial wall digested
Centrifuged(peeled) protoplast
Isolatedprotoplasts
Plating of protoplast
Wall regeneration
First division
Clump of cells
Colony formation
Callus
Callus differentiation
Regenerated plantlet
Young plant

23.  The lower surface of the sterilized leaf is peeled off and
stripped leaves are cut into pieces (midrib). The peeled leaf
segments are treated with enzymes (macerozyme and then
treated with cellulase) to isolate the protoplasts.
 The protoplasts so obtained are cleaned by centrifugation
and decantation method. Finally, the protoplast solution of
known density (1 × 105 protoplasts/ml) is poured on sterile
and cooled down molten nutrient medium in petridishes. Mix
the two gently but quickly by rotating each petridish. Allow
the medium to set and seal petridishes with paraf-fin film.
Incubate the petridishes in inverted position in BOD
incubator. The protoplasts, which are capable of dividing
undergo cell divisions and form callus within 2–3 weeks.
 The callus is then sub-cultured on fresh medium.
Embryogenesis begins from callus when it is transferred to a
medium containing proper proportion of auxin and cytokinin,
where the embryos develop into plantlets which may be
transferred to pots

24. Hairy Root Culture
 The name ‘hairy root’ was mentioned in the literature by
Steward et al. (1900).
 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.
 A diversified range of plant species has been
transformed using various bacterial
 strains.

25.  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
(a) (b)
Hairy root culture of Vinca: (a) in solid media and (b) in liquid media

26. ESTABLISHMENT AND MAINTENANCE
OF VARIOUS CULTURES
 Growth of callus masses on solidified media (callus
culture also known as static culture).
 Growth in liquid media (suspension culture) consists of
mixture of single cells or cell aggregates.
 Protoplast culture:
 Callus culture (static tissue culture) or
 Suspension culture

27. 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; unlike tumor tissue, the cell division takes
place periclinally.
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.

28. PREPARATIO
N:
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 min. In the
case of plant organ containing waxy layer, the
material is either pretreated with wetting agents
[ethanol 70–90%; tween 20 (polyoxyethylene
sorbitan monolaurate): 1–20 drops into 100 ml
distilled water]; 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).

29. (B) SELECTION OF CULTURE
MEDIUM
 The organ is to be cultured in well-defined nutrient
medium containing inorganic and organic nutrients and
vitamins.
 The culture of the 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, 1BA and NAA are widely used in
medium for rooting and in combination with cytokinin
for shoot proliferation.

30. (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 autocontrolled 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. Many tissue explants possess some degree of
polarity with the result that the callus is formed most early
at one surface. In stem segment, callus is formed
particularly from that surface which in vivo is directed
towards the root.

31. Callus is formed through three stages of development,
such as:
 Induction
 Cell division and
 Cell differentiation
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.
Cell division
 This is the phase of active cell division as the explant
cells revert to meristematic state.

32. Cell differentiation
 This is the phase of cellular differentiation, i.e.
morphological and physiological differentiation occur
leading to the formation of secondary metabolites.
Suspension Culture
 Suspension culture contains a uniform suspension of
separate cells in liquid medium. For the preparation of
suspension culture, callus fragments is 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, subculturing can be done in
fresh liquid medium. Single cellscan also be obtained from
fresh plant organ (leaf).

33. 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 60ml of liquid nutrient
media (i.e. defined nutrient medium without gelling
agent), the flasks 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.

34. (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
 Enzymatic method
 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 (two layers) cloth,
washed with sterile distilled water, centrifuged
with culture medium, sieved and placed on
culture dish for inoculation.

35.  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 (2 g leaves) 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.

36. APPLICATIONS OF PLANT TISSUE
CULTURE
 Plant tissue culture technology has been used in almost
all the field of biosciences. The desirable products
produced by plant tissue cultures are as diversified as is
industry itself.
 Its applications include:
 Production of Phytopharmaceuticals
 Biochemical Conversions
 Clonal Propagation (Micro-propagation)
 Production of Immobilized Plant Cells

37.  Production of Phytopharmaceuticals
 The use of plant tissue culture for the production of
phytopharmaceuticals was started in 1959 when
Wenstein et al., studied Agave, for the production of
steroids using tissue culture method.
 Dioscorea was reported to contain industrially useful
steroids by 1966, but it was 1969, when Kaul reported the
production of 1.2% dry weight diosgenin by tissue culture
of D. sylvatica.
 During the last two decades advancement in tissue
culture technology such as development of hairy root
cultures, immobilized plant cell systems, and technique to
enhance the excretion of desired product into medium has
resulted in promising findings for a variety of
medicinallyimportant substance from several medicinal
plants.

38. PRODUCTION OF
PHYTOPHARMACEUTICALS
 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 isomenthone.

39.  Podophyllum peltatum in semicontinuous culture
can produce anticancer drugs by bio-transformation
of synthetic dibenzyl butanolides to lignans—
suitable for conversion to etoposide.
 In some tissue culture stereospecific bio-
transformation is also reported, which is important
for the isolation of optically active compound from
racemic mixture.
 Example of cell culture of Nicotiana tabacum
selectively hydrolyses R-configurational form of
monoterpenes like bornyl acetate and isobornyl
acetate.
 Apart from the above-mentioned biochemical
conversions, many other, like saponifications,
esterification, epoxidation, oxidation, methylation
and isomerization are also reported

40.  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.

41.  Somaclonal Variation
 In clonal propagation, clones are produced from tissue
culture with uniform characters but few clones may show
variations among the population of clones, which were not
present in the parent cells. This formation of variant
clones from cultured tissue is called as somaclonal
variations.
 Variants are of two types:
(i) desirable variants and
(ii) undesirable variants.
 Desirable variants can be used for the improvement of
crops. The clone showing high productivity can be used for
commercial purposes.

42.  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.

43.  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.
 Subsequently, the cultures convert anisodamine into
scopolamine.
 The bio-transformation reactions, such as glycosylations,
hydroxylation, acetylation, demethylation, etc., have been
successfully attempted in immobilized cell systems.
 The hydroxylation or glycosylation of cardiac glycosides in
cultures of Digitalis lanata and Daucus carota have also
been reported.

44.  Immobilized plant cells can be used for tracing
the biosynthetic pathways of secondary
metabolites and also can be used for carrying out
bio-transformation or biochemical reactions.

45. REFERENCE
TEXTBOOK OF
PHARMACOGNOSY
AND PHYTOCHEMISTRY
By
Biren N. Shah & A.K. Seth