Plant tissue culture

PLANT TISSUE CULTURE
PREPARED BY
MRS. MEGHA S SHAH
ASSISTANT PROFESSOR, DEPARTMENT OF PAHARMACOGNOSY
AISSMS COLLEGE OF PHARMACY, PUNE
1

DEFINATION AND HISTORY
OF PLANT TISSUE CULTURE
2

DEFINATION
Plant-tissue culture is in-vitro cultivation of plant cell or tissue under aseptic and
controlled environment conditions, in liquid or on semisolid well defined nutrient
medium for the production of primary and secondary metabolites or to regenerate
plant.
In other words it is an experimental technique through which a mass of cells
(callus) is produced from an explants tissue.
The callus produced through this process can be utilized directly to regenerate
plantlets or to extract or manipulate some primary and secondary metabolites.
3

Historical development of plant tissue culture
The principle of tissue culture were evolved 1838-1839 in cell theory advanced by Schleiden and
Schwann.
4
1902 Haberblandt proposed concept of in vitro cell culture
1904 Hanning Culture embryos from several cruciferous species.
1922 Kotte and Robbins successfully cultured root and stem respectively
1926 Discovered first plat growth hormone Indole acetic acid (IAA)
1934 White introduced Vitamin B as growth supplement in tissue culture
media for tomato root tip.
1939 Gautheret, white and Nobecourt established Endless proliferation of
callus culture.

1941 Van Overbeek was first to add coconut milk for development of embryo and
callus formation (cell division) in Datura
1946 Ball raised whole plants of Lupinus by shoot tip culture
1954 Muir was first to break callus tissues into single cells
1957 Skoog and Miller gave concept of Hormonal Contol (auxin: cytokinin) on shoot
and root induction in tobacco callus culture.
1966-1968 Reinert and steward regenerated embroys from callus clumps and cell
suspension of Daucus carota.
1972 Carlson Produced first somatic hybrid plant by fusing the protoplast of
Nicotiana glauca and N. Langsdorfii
1981 Vilnken suceeed in electrical fusion of protoplast
5

ADVANTAGES AND DISADVANTAGES
OF PLANT TISSUE CULTURE
6

Advantages of tissue culture
1. Availability of raw material: Some plants are difficult to cultivate and are also not available
in abundance and tissue culture technique is considered a better source for regular and uniform
supply of raw material for medicinal plant industry for production of phytopharmaceuticals.
2. Fluctuation in supplies and quality The method of production of crude drugs is variable in
quality due to changes in climate, crop diseases and seasons. All these problems can be
overcome by tissue culture.
3. New methods for isolation: It is possible to obtain new methods for isolation and newer
compounds from plant by this technique and for which Patent rights can be obtained.
7

4. Biotransformation: (Process through which the functional group of organic compound are modified by
living cells) reactions are feasible using plant-cell cultures.
5. Disease free and desired propagule : Large scale production of plant with disease free and desired
propagule could be stored and maintained without any damage during transportation for subsequent
plantation.
6. Biosynthetic pathway: Tissue culture can be used for tracing the biosynthetic pathways of secondary
metabolites using labelled precursor in the culture medium.
7. Immobilization of cells: Tissue culture can be used for plants preservation by immobilization
(entrapment) of cell further facilitating transportation and biotransformation.
8

8. Continuous, uniform biomass is obtained.
9. Medicinally important compound can be synthesized, which can’t
be synthesized chemically.
10. Useful natural compounds can be produced, independent of soil condition & change in
climatic conditions.
11. Improvement of medicinal plant species.
12. Propagation of plant without seeds in defined and controlled condition.
9

Disadvantages of tissue culture
1. High level of expertise is required.
2. A small error may lead to complete collapse of product/plant.
3. Lots of chemicals are required for plant tissue culture which must contain high purity.
4. There is no chance for evaluation of mutation.
5. Culture on artificial medium may lead to the depression of unusual metabolic pathways,
which may not be beneficial to biotechnologist.
6. In majority cases amount of secondary metabolites produced is negligible.
10

Disadvantages of tissue culture
7. The protocols for individual plants differ very widely and Change in the medium constitution &
environmental parameters affect the rate of cell growth & accumulation of secondary metabolites.
8. To maximize on the cell mass produced the cell suspension culture eventually becomes very dense and
these presents problems of even aeration.
9. Instability
10. Slow growth
11. Expensive process
12. Aseptic conditions are to be maintained through out the growth of plant.
11

LABORATORY REQUIREMENTS AND
GENERAL PROCEDURE OF PLANT
TISSUE CULTURE
12

Laboratory Requirements Of Plant Tissue Culture
A. Laboratory requirements for plant tissue culture
1. Washing, drying and storage of vessels
2. Preparation, sterilization and storage of media
3. Aseptic handling of explants and cultures
4. Maintenance of culture
5. Observation of Cultures
1 and 2 are done in media room. 3 to 5 are performed in culture room.
13

The working area should be physically separated, even by a temporary partition from that
used for medium preparations.
The weighing balance should be kept in separate space.
Refrigerator, deep freeze, incubators and autoclave may be kept in corridor.
For aseptic manipulation, Laminar flow hoods are commonly used which can be housed in
culture room.
A small table having stereoscopic microscope may be adequate for culture observation.
14

B. Culture room
Controlled temperature
Culture racks fitted with light (generally 1000 lux or lower power light generated by
fluorescent tubes)
A shaker for agitation of liquid medium.
Providing generator set for providing electricity to the culture room when there is
electricity failure or cuts.
15

C. Culture vessels and their washing
Glass culture vessel are used : Cheaper, reusable and autoclavable
To use only borosilicate or Pyrex glass. No ordinary soda glass (may be toxic to some tissues)
Culture vessel of plastic are available for variety of purpose, they are presterilized and
disposable, but certain types are autoclavable and therefore reusable.
But plastic vessels in the long run are costly than glass vessels.
Tissue are culture in Culture tubes, Flasks and petridish but mainly specially designed dishes
are used also used.
16

Wide mouth bottles, including milk bottles are often employed especially for micropropagation work.
Suspension culture of necessity are maintained in long neck culture flasks.
Culture tubes and flasks are usually stopper with cotton plugs, which are often wrapped by muslin cloth, but
preparaing such plugs on large scale may be time consuming and inconvenient.
Use caps made up of either polypropylene or metal for this purpose. Caps are effective but their design may affect
their performance.
Culture vessel and other lab ware are generally soaked suitable nontoxic detergent solution overnight, washed with
suitable brush, thoroughly rinsed, clean with tap water, followed by rinse with distilled water and dried in hot air
oven (75 to 80 0C)
Culture vessels having contamination are first autoclaved. Washed culture vessels should be washed stored preferably
in dust Proof cabinet.
17

D. Sterilization
(i) Dry Heat: In hot air Oven at 160-1800C for 3 hr. But mostly prefer autoclave.
(ii) Flame sterilization: Instruments like Scalpel, needles, forceps are sterilized by this
method.
(iii) Autoclaving: Culture vessel both empty and media containing are sterilized.
(iv) Filter sterilization: certain vitamins, enzymes, growth regulators are heat liable and
sterilized by this technique using membrane filter of 0.45 micron or lower pore size.
(v) Wiping with 70 % Ethanol: Surface of any instruments and platform are sterilized by
wiping with 70% alcohol and then allowed to dry.
(vi) Surface sterilization: for a.ll plant material
18

Preparation of Explants
Explants can be defined as a portion of plant body, which has been taken from the plant to establish
a culture.
Explants 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.
Explants may be taken from any part of the plant like root, stem, leaf, or meristematic tissue like
cambium, floral parts like anthers, stamens, etc.
Age of the explants 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.
When seeds and grains are used for explants preparation, they are directly sterilized and put in
nutrient medium. After germination, the obtained seedlings are to be used for explants preparation.
19

Surface Sterilization of Explant
For the surface sterilization of the explant, chromic acid, mercuric chloride (0.11%), calcium
hypochlorite, sodium hypochlorite (1–2%) and alcohol (70%) are used.
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.
In the case of leaf or green fresh stem the explants 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 (due to waxy secretion).
20

Name of Chemical Concentration (%) Exposure (min)
Bromine water 1–2 2–10
Benzalkonium chloride 0.01–0.1 5–20
Sodium hypochlorite 0.5–5 1 5–30
Calcium hypochlorite 9–10 5–30
Mercuric chloride 1–2 2–10
Hydrogen peroxide 3–10 5–15
Silver nitrate 1–2 5–20
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Surface sterilizing agent

Procedure to be followed for respective explants is as follow:
Seeds
1st Step: Dip the seeds into absolute ethyl alcohol for 10 s and rinse with purified water.
2nd Step: Expose seeds for 20–30 min to 10% w/v aqueous calcium hypochlorite or for 5 min in
a 1% solution of bromine water.
3rd Step: Wash the treated seeds with sterile water (three to five times) followed by germination
on damp sterile filter paper.
Fruits
1st Step: Rinse the fruit with absolute alcohol.
2nd Step: Submerge into 2% (w/v) solution sodium hypochlorite for 10 min.
3rd Step: Washing repeated with sterile water and remove seeds of interior tissue.
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Stem
1st Step: Clean the explant with running tap water followed by rinsing with pure alcohol.
2nd Step: Submerge in 2% (w/v) sodium hypochlorite solution for 15–30 min.
3rd Step: Wash three times with sterile water.
Leaves
1st Step : Clean the leaf explants with purified water to make it free from dirt and rub the
surface with absolute ethyl alcohol.
2nd Step: Dip the explants in 0.1% (w/v) mercuric chloride solution.
3rd Step: wash with sterile water to make it free from chloride and then dry the surface with
sterile tissue paper.
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Production of Callus from Explants
The sterilized explants is transferred aseptically onto defined medium contained in flasks. The flasks are
transferred to BOD incubator for maintenance of culture.
Temperature is adjusted to 25±2oC. Some amount of light is necessary for callus (undifferentiated
amorphous cell mass) production.
Usually sufficient amount of callus is produced within three to eight days of incubation.
Proliferation of Callus
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.
24

Sub-culturing of Callus
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
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.
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.
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NUTRIENT MEDIA FOR PLANT TISSUE
CULTURES/ MEDIA COMPOSITION
26

To maintain the vital functions of a culture, the basic medium consisting of inorganic nutrients
(macronutrients and micronutrients) adapted to the requirements of the object in question, must be
supplemented with organic components (amino acids, vitamins), growth regulators (phytohormones) and
utilizable carbon (sugar) source and a gelling agent (agar/phytogel).
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(s) in culture.
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Macronutrients: The macronutrients include six major elements: nitrogen (N),
phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg) and sulphur (S)
present as salts that constitute the various above mentioned defined media.
The concentration of the major elements like calcium, phosphorus, sulphur and
magnesium should be in the range of 1–3 mmol l-1 where as the nitrogen in the media
(contributed by both nitrate and ammonia) should be 2–20 mmol l-1.
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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.
29

The composition of some plant tissue culture media reveal that the chief difference in the composition of
various commonly used tissue culture media lies in the quantity of various salts and ions.
Qualitatively, the inorganic nutrients required for various culture media appear to be fairly constant.
The active factor in the medium is the ions of different types rather than the salt (mineral salts on
dissolving in water undergo dissociation and ionization).
A single ion may be contributed by more than one salt.
For example, in Murashige and Skoog’s medium, NO3 – ions are contributed by NH4NO3 as well as
KNO3 and K+ ions are contributed by KNO3 and KH2PO4.
31

White’s medium, one of the earliest plant tissue culture media, includes all the necessary
nutrients and was widely used for root culture.
The experience of various investigators has however revealed that quantitatively the
inorganic nutrients are inadequate for good callus growth (Murashige and Skoog’s, 1962);
hence, most plant tissue culture media that are now being widely used are richer in mineral
salts (ions) as compared to White’s medium.
Aluminium and nickel used by Heller’s (1953) could not be proved to be essential and,
therefore, were dropped by subsequent workers, but sodium, chloride and iodide are
indispensable.
32

In Heller medium, special emphasis was given to iron and nitrogen. In the original White’s medium iron was
used in the form of Fe2(SO4), but Street and coworkers replaced it by FeCl3 for root culture because of the
impurities due to Mn and some other metallic ions.
However, FeCl3 also did not prove to be an entirely satisfactory source of iron.
In this form iron is available to the tissue culture at or around pH 5.2 and within a week of inoculation the pH
of the medium drift from 4.9–5.0 to 5.8–6.0, and the root culture started showing the iron deficiency
symptoms.
To overcome this difficulty, in most medium, iron is now used as FeEDTA; in this form, iron remains
available up to a pH of 7.6–8.0.
However, unlike root, callus cultures can utilize FeCl3 to pH 6.0 by secreting natural chelates. FeEDTA may
be prepared by using Fe2(SO4)37H2O and Na2EDTA 2H2O.
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Organic nutrients
Nitrogenous substances:
Most cultured plant cells are capable of synthesising essential vitamins but not in sufficient
amount. To achieve best growth it is essential to supplement 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
pentothenate (vitamin B5) and ionositol are also known to improve growth of the tissue
culture material.
There is variation in the quantities of essential vitamins used by various standard media.
34

Numerous complex nutritive mixtures of undefined composition, like casein hydrolysate,
coconut milk, corn milk, malt extract, tomato juice and yeast extract have also been used to
promote growth of the tissue culture, but these substances specifically fruit extracts may affect
the reproducibility of results because of variation in the quality and quantity of growth
promoting constituent in these extracts.
Carbon Source:
It is essential to supplement the tissue culture media with an utilizable source of carbon to the
culture media. Haberblandt (1902) attempted to culture green mesophyll cells, probably with
the idea that green cells would have simple nutritive requirement, but this did not prove to be
true.
In fact even fully organized green shoot in cultures, and it also did not show proper growth and
proliferation without the addition of suitable carbon source in the medium.
35

The most commonly used carbon source is sucrose at a concentration of 2–5%.
Glucose and fructose are also known to be used for good growth of some tissues. Ball (1953, 1955)
demonstrated that 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 where as monocots do best with
dextrose (glucose).
Some other forms of carbon that plant tissues are known to utilize include maltose, galactose, mannose,
lactose and sorbitol. It has been reported that some tissues can even metabolize starch as the sole carbon
source, e.g. tissue cultures of sequoia and maize endosperm.
36

Plant growth regulators
Plant growth regulators are the critical media components in determining the developmental pathway of
the plant cells. The plant growth regulators used most commonly are plant hormones or their synthetic
analogues.
Auxins
Auxins promote both cell division and cell growth.
The most important naturally occurring auxin is IAA, 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 to partially alleviate the problems associated with the use of
IAA. It is more common, though, to use stable chemical analogues of IAA as a source of auxin in plant
cell culture media.
2,4-Dichlorophenoxyacetic acid(2,4-D) is the most commonly used auxin, and is extremely effective in
most circumstances.
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Cytokinins
Cytokinins promote cell division. Naturally occurring cytokinins are a large group of structurally
related(they are purine derivatives) compounds of the naturally occurring cytokinins, two have some use
in plant tissue culture media.
These 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 (benzylaminopurine), are therefore used more frequently.
Nonpurine based chemicals, such as substituted phenylureas, are also used as cytokinins in plant cell
culture media. These substituted phenylureas can also substitute for auxin in some culture systems.
Abscisic acid: ABA inhibits cell division. It is most commonly used in plant tissue culture to promote
distinct developmental pathways such as somatic embryogenesis.
38

Gibberellins: There are numerous, naturally occurring, structurally related compounds termed 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.
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. It
does, though, present a particular problem for plant tissue culture.
Some plant cell cultures produce ethylene, which, if it builds up sufficiently, can inhibit the growth and
development of the culture.
The type of culture vessel used and its means of closure affect the gaseous exchange between the culture
vessel and the outside atmosphere and thus the levels of ethylene present in the culture.
39

Solidifying agents/ gelling agent 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.
Most commonly used substance for this purpose is the phycocolloid agar–agar obtained from red
algae (Gelidium gracilaria).
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.
However, agar is not an essential component of the nutrient medium. Single cell and cell aggregates
can be grown as suspension cultures in liquid medium containing inorganic, organic nutrients and
other growth factors. Such culture should however be regularly aerated either by bubbling sterile air
or gentle agitation.
40

In nutritional studies, the use of agar should be avoided because of the impurities present in all the
commercially available agar–agar especially of Ca, Mg, K, Na and trace elements.
Agar (Agarose) is extraordinary resistant to enzymatic hydrolysis at incubation temperature, and only a
few bacteria exist which are capable of producing degrading enzyme— agarose.
This resistance to hydrolysis is the fundamental importance to the use of agar–agar in cell culture
medium.
It is also neutral to media constituents and thus do not react with them.
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.
41

Media Preparation
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.
Solution preparation
For the preparation of stock solution, each component (analar grade) 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-1 or 10 mmol l-1. All the stock solutions
are stored in refrigerator till used.
For iron solution, dissolve FeSO4 7H2O and Na2EDT.2H2O separately in about 450 ml distlled
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.
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Semisolid media preparation
Required quantities of agar and sucrose are weighed and dissolved in water by 3/4th volume of
medium, by heating them on water bath. Adequate quantities of stock solution (for 1L medium
50 ml of stock solution of Group 1, 5 ml of stock solution II, III and IV group) 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 aluminium foils and are autoclaved at pressure of 2–2.2 atm at 121°C for 15–40 min (time
to be fixed from the time when temperature reaches the required temperature).
The exposure time depends on the volume of the liquid to be sterilized as given below
43

Minimum autoclaving time includes the time
require for the liquid volume to reach the
sterilizing temperature (121°C) and 15 min at
this temperature.
Time may vary due to difference in autoclaves.
Moreover, the actual success of sterilization
can be tested using A bio-indicator, commonly
spores of the bacterium bacillus
stearothermophillus 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 nonautoclaved controls
are incubated For 24–48 h at 60°C.
If the spores are dead, the color Of the pH
indicator in the Solution remains unchanged
indicating no change in pH.
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Volume of the media per
vessel (ml)
Minimum autoclaving
time (min)
25 20
50 25
100 28
250 31
500 35
1000 40
2000 48
4000 63

1. Depending upon the type of medium
46
culture
Suspension
culture
Batch culture
Semicontinuous
culture
Continuous
culture
Open type
chemostats
turbidostats
Close type
Callus (static
culture)

2. Depending on the part used for culture
a) Organ Culture
i. Root tip culture
ii. Shoot tip culture
iii. Leaves or leaf primordia culture
iv. Complete Flower Culture
v. Anther and Pollens Culture
vi. Ovule and Embryo Culture
vii. Seed Culture
b) Hairy Root Culture
c) Protoplast Culture and Somatic Hybridization
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i. Root tip culture (Meristem – 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 radical could be multiplied and
maintained in continuous culture; such genetically uniform root cultures are
referred to as a clone of isolated roots.
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ii. Shoot-tip culture (Meristem – 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.
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iii. 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.
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iv: 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.
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v. 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.
52

Mature anther or pollen grains (microspora) 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.
53

vi. 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.
54

vii. Seed Culture
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.
55

Protoplast Culture
Protoplasts are the naked cells of varied origin without cell walls, which are cultivated in
liquid as well as on 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.
56

The lower surface of the sterilized leaf is peeled off and stripped leaves are cut into pieces
(midrib).
Protoplast are isolated by two method
1) Mechanical method: In this method the plasmolysed cells (infact cell wall) are cut with
sharp knife to release the protoplasts. This method gives poor yield of protoplasts thus it
is no more practically used.
2) Enzymatic method: The peeled leaf segments are treated with enzymes (macerozyme and
then treated with cellulase) to isolate the protoplasts.
58

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 paraffin film.
Incubate the petridishes in inverted position in BOD incubator.
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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 embroys
develop into plantlets which may be transferred to pots.
60

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 vitamin and
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.
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.
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In the production of hairy root cultures, the
explant material is inoculated with a
suspension of Agrobacterium rhizogenes.
The bacteria contains root inducing plasmid
(Ri).
This culture is generated by growing bacteria
in yeast maltose broth (YMB) medium for 48
hr at 250 C with rotating shaking, pelleting by
centrifugation and resuspending the bacteria
in YMB medium to form a thick suspension.
62

Transformation may be induced on aseptic plants grown from seed or on detached leaves, leaf
disc, petioles or stem segments from green house plants followed by sterilization of the excised
tissues.
In some species a profusion of root may appear directly at the site of inoculation, but in others a
callus will form initially and root emerge subsequently from it.
In either case, hairy roots appear within one to four weeks.
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.
63

Callus Culture (Static 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
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(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 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–
5mm).
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(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, 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. This is also quite favorable for monocot tissues or explant.
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The selected semisolid nutrient is prepared. The pH of the medium is adjusted
(5.0–6.0) and poured into culture vessels (15 ml for 25 x 150 mm culture tubes or
50 for 150 ml flasks) plugged and sterilized by autoclaving.
(c) Transfer of explants
Surface sterilized organs (explants) from stem, root or tuber or leaf, etc., are
transferred aseptically into the vessel containing semisolid culture medium.
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(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 explants.
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.
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The unique feature of callus is its ability to develop normal root and shoot, ultimately forming a
plant.
Commercially important secondary metabolites can also be obtained from static culture by
manipulating the composition of media and growth regulators (physiological and biochemical
conditions), but on the whole it is a good source for the establishment of suspension culture.
Callus is formed through three stages of development, such as
Induction
Cell division and
Cell differentiation
69

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 explants and the environmental
conditions of the cell division stage.
Cell division
This is the phase of active cell division as the explants cells revert to meristematic state.
Cell differentiation
This is the phase of cellular differentiation, i.e. morphological and physiological
differentiation occur leading to the formation of secondary metabolites.
70

Maintenance
After sufficient time of callus growth on the same medium following change will occur, i.e.
Depletion of nutrients in the medium
Gradual loss of water
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.
71

Callus tissue may appear in the following different colors.
White: If grown in dark due to the absence of chlorophyll
Green: If grown in light
Yellow: Due to development of carotenoid pigments in greater amounts.
Purple: Due to the accumulation of anthocyanins in vacuole.
Brown: Due to excretion of phenolic substance and formation of quinones.
Callus culture may vary widely in texture appearance and rate of growth. Some callus growth is
heavily lignified and hard in texture while others are fragile. The cells in callus tissue vary in shape
from spherical to elongated.
72

Suspension Culture
Suspension culture contains a uniform suspension of separate cells in liquid medium.
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 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.
73

(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 (two layers) cloth, washed with sterile
distilled water, centrifuged with culture medium, sieved and placed on culture dish for
inoculation.
74

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.
75

Growth pattern of suspension culture
The cell number of a biomass of a batch culture
exhibit a typical sigmoidal curve.
having a lag phase during which the cell number
of biomass remains unchanged,
followed by a logarithamic (log) phase
(Exponential phase) when there is rapid increase
in cell number
finally ending in a stationary phase during
which cell number gradually declines.
76

The incubation period from culture initiation to the stationary phase is determined primarily by
(a) Initial cell density
(b) Duration of lag phase and
(c) Growth rate of cell type
The cell density used to sub-culture is critical and depend largely on the type of suspension culture
to be maintained.
The low initial cell density will prolong the lag phase and exponential phase of growth. At an initial
cell density of 9–15 × 103 ml, the cell will generally undergo eightfold increases in cell number
before entering the stationary phase. Normal incubation time of stock culture is 21–28 days, while
for sub-culture it is 14–21 days.
77

Parameters for measuring growth of cultured cells
1. Fresh weight: The value of callus cultures, frequently determined as total weight of callus
medium layer and petridish. However, in this method, there are variations due to evaporation
via the medium’s surface. Hence, 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 separate layers of cellulose or nylon.
2. Dry weight: It requires repeated drying usually at 60°C to the point of constant weight, up to
fresh weight of 500 mg, a linear relationship between fresh and dry weight is assumed. This
method excludes error due to varying endogenous water contents.
78

3. Cell mass: It may be determined by densification by centrifugation (Ca 2000 g, 5 min) of a
particular percentage of the volume (4–7 ml) in graduated conical centrifuge tubes. In order to
avoid error, due to water absorption by the cells, the so-called packed cell volume (PCV) must
be recorded immediately following the separation process.
4. Cell number: To determine the number of cell per unit volume, existing cell clumps or
aggregates must be separated into isolated cell (callus culture and in most suspension cultures).
This is commonly done using chrome-trioxide alone or in combination with hypochlorous acid.
Possible alternative are EDTA and pectinase.
79

5. 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 concentration.
As long as the pH of the medium remains above 3, the concentration of hydrogen ions does not affect
conductivity.
6. Cellulose concentration: Calcofluor-white ST (0.1% aqueous) allows monitoring of changes in the
concentration of cell wall polymers from β-glucoside bond glucose molecule such as cellulose or
callose. The textile brightener specifically bonds to β-1,4 glucans and intensely fluoresces following
stimulation with short wave blue light. In this way even traces of these compounds may be identified.
80

Maintenance of suspension culture
(i) Batch suspension culture:
In this technique, the cells are allowed to multiply in liquid medium, which is continuously
agitated to break up cell aggregates.
The system is closed with respect to additions or removal of culture, except for circulation of
air.
In this technique to commence the growth again on the 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.
81

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.
(ii) 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.
82

(iii) 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.
83

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.
In chemostat, the desired rate of growth is maintained by adjusting the level of concentration of
nutrient by constant inflow of fresh medium.
In turbidostat, on the contrary, the input of medium is intermittent, and it is mainly required to
maintain the cell density in the culture.
84

APPLICATION OF PLANT
TISSUE CULTURE
85

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 (secondary metabolites)
Biochemical Conversions (Biotransformation)
Clonal Propagation (Micro-propagation)
Production of Immobilized Plant Cells (cell immobilization)
86

Production of Phytopharmaceuticals
Secondary plant
metabolites like
alkaloids, terpenoids,
flavonoids, lipids,
oils, tannins,
anthraquinones,
flavones,
napthaquinones,
vitamins, proteins,
anticancer agents,
antiviral agents etc.
are isolated from
plant tissue culture.
Compound Plant Species Culture type
Ajmalicine Catharanthus roseus suspension
Atropine Atropa belladonna Hairy root culture
Berberine Coptis japonica Callus and suspension
Caffeine Coffea arabica callus
Cardenolides Digitalis purpurea
Digitalis lanata
Callus and suspension
S (Biotransformation)
Codeine Papaver somniferum suspension
Glycyrrhizin Glycyrriza glabra suspension
Morphine Papaver somniferum suspension
Nicotine Nicotiana tobacum suspension
Papain Carcia papaya callus
87

Biochemical Conversions (Biotransformation)
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.
88

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.
89

Clonal Propagation (Micro-Propagation)
Clonal propagation (micro-propagation) is the technique to produce entire plant from
single individual by asexual reproduction.
It is 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.
90

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.
91

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.
92

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.
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.
93

The following methods are used for the immobilization of plant cells:
(a) gel entrapment
The most common method used for plant cell immobilization is gel entrapment and the most
common gel used is calcium alginate.
A mixture of sodium alginate and cell is extruded drop-wise into a calcium chloride solution,
where bead hardening starts to take place immediately.
Alginate is the preferred gel because of the ease with which it can be handled and because of its
mildness. Moreover, calcium alginate can have beneficial effects on the production of
secondary metabolites.
Asada and Shuler (1989) immobilized Catharnthus roseus cells in calcium alginate, which
resulted in a three-fold increase in extra-cellular ajmalicine.
94

(b)Biofilms
Another application of gels for the immobilization of plant cells is their use as a support for
biofilm formation.
Krgi (1988) grew Catharanthus roseus cells on the surface of calcium alginate beads within the
interspatial volume of a packed column. The cells showed some growth and the alkaloid content
of the medium were increased.
(c)Adsorption
Several authors immobilized plant cells by adsorption to various support materials, such as fiber
glass, polystyrene, sulfonated polystyrene, fluorinated ether etc
(d)Foam immobilization
Another material that has found widespread use in plant cell immobilization is polyurethane
foam.
95

Production of Secondary metabolites in immobilized cell
96
Species Product Increase (X fold)
Capsicum frutescens Capsaicin > 100
Capsicum annum Capsaicin > 100
Coffee arabica Methyl xanthenes 13
Catharanthus Roseus Ajmalacine 25

selected one step bioconversion by immobilized cells
97
Cell culture species Reaction type Precursor Product Matrix
Digilatis lanata Hydroxylation β-methyl
digitoxin
β-methyl digoxin Alginate
Daucus Lanata Hydroxylation Digitoxigenin Periplogenin Alginate
Mentha Species Reduction (-) Menthone (+) neomenthol PAAH
Papaver somniferum Reduction Codinone Codeine Alginate & PUR

EDIBLE VACCINE
Edible vaccines are transgenic plant and animal based production of or those that contain agents that
trigger an animals immune response. In simple terms edible vaccines are plant or animal made
pharmaceuticals.
Edible vaccine contain DNA fragments from the original pathogen. These fragments code for a protein
that is usually a surface protein of the pathogen. This is responsible for eliciting the body’s immune
response.
The concept of edible vaccines was developed by Arntzen in 1990s. Although this idea seemed quite
simple in the beginning but making it into reality has required sophisticated science. The earliest
demonstration of an edible vaccine was the expression of a surface antigen from the bacterium
Streptococcus mutans in tobacco.
99

Advantage of edible vaccine
They are cheap so they can be produced in large.
They can be ingested by eating the plant/part of the plant. So the need to process
and purify does not arise.
Extensive storage facilities like cold storage are not required.
Most importantly, they trigger the immunity at the mucosal surfaces such as
those that line the mouth (mucosal immunity) which is body’s first line of defense.
101

Disadvantages of edible vaccines
There is question mark in the survival of antigen in the acidic conditions of the stomach and
if they did will they be able to trigger the immune system in right way. Although initial trial
have shown promising results in human subjects but it is not clear what will happen when
the person comes in contact with actual virus.
To control the dose of vaccine is the most difficult task. There seems to be danger that too
high dose could provoke oral tolerance of an invading bacteria or virus instead of an
immune response. Also the dosage requirements for children and adults will be different.
Plants are living organism that change, so the continuity of the vaccine production might
not be guaranteed.
People may develop an allergy to the fruits or vegetable expressing the foreign antigen.
Glycosylation patterns in plants differ from those in humans and could affect the
functionality of vaccines.
102

QUESTION BANK
1. Enumerate name of types of tissue culture. 2 marks
2. List out the application of plant tissue culture 2 marks
3. Note on Hairy root Culture. 5 marks
4. Laboratory requirements for plant tissue culture. 5 marks
5. Describe types of tissue culture based on plant of parts used. 5 marks
103

QUESTION BANK
6. Advantages and Disadvantages of plant tissue culture. 5 marks
7. Write a note on Edible vaccine. 5 marks
8. Briefly describe Suspension culture. 5 marks
9. Explain Protoplast Culture. 5 marks
10. Note on Cell immobilization technique by suitable example. 5 marks
11. Nutritional requirements for plant tissue culture. 10 marks
12. Application of plant tissue culture technique. 10 marks
104

Plant tissue culture

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