PLANT TISSUE CULTURE

Introduction- 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 metabolite 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 and amino acids as
well as sterilization of explants, glassware and other accessories inoculation and
incubation.
Advantages – 1) Availability of raw material
2) Fluctuation in supplies and quality
3) Patent right
4) Political reasons
5) Easy purification of the compound
6) Modification in chemical structure
7) Disease-free and desired propagule
8) Crop improvement
9) Biosynthetic pathway
10) Immobilization of cells

Availability of raw material- some plants are difficult to cultivate and are also not
available in abundance. In such case, the biochemical/bioproducts from these plants
cannot be obtained economically in sufficient quantity. So tissue culture is considered a
better source for regular and uniform supply of raw material, manageable under regulated
and reproducible conditions in the medicinal plants industry for the production of
phytopharmaceuticals.
Fluctuation in supplies and quality- The production of crude drugs is subject to
variation in quality due to changes in climate, crop diseases and seasons. The method of
collection, drying and storing also influence the quality of crude drug. All these problems
can be overcome by tissue culture techniques.
Patent rights- Naturally occurring plants or their metabolites cannot be patented as such.
Only a novel method of isolation can be patented. For Rand D purpose, the industry has
to spend a lot of money and time to launch a new natural product but can’t have patent
right. Hence, industries prefer tissue culture for production of biochemical compounds.
By this method ,it is possible to obtain a constant supply and new methods can be
developed for isolation and improvement of yield , which can be patented.
Political reasons-if a natural drug is successfully marketed in a particular country of its
origin, the government may prohibit its export to up-value its own exports by supplying
its phytochemical product, e.g. Rauwolfia serpentina and dioscorea spp.from India.
Similarly the production of opium in the world is governed by as such by political
consideration, in such case, if work is going on the same drug; it will be either hindered
or stopped. Here also, plant tissue culture is the solution.
Easy purification of the compound- The natural products from plant tissue culture may
be easily purified because of the absence of significant amounts of pigments and other
unwanted impurities. With the advancement of modern technology in plant tissue culture,
it is also possible to biosynthesize those chemical compounds which are difficult or
impossible to synthesize.
Modification in chemical structure- some specific compound can be achieved more
easily in cultured plant cells rather than by chemical synthesis or by microorganism.
Disease-free and desired propagule- plant tissue culture is advantageous over
conventional method of propagation in large scale production of disease free and desired
propagules in limited space and also the germplasm could be stored and maintained
without any damage during transportation for subsequent plantation.
Crop improvement-Plant tissue culture is advantageous over the conventional
cultivation techniques in crop improvement by somatic hybridization or by production of
hybrids.
Biosynthetic pathway- Tissue culture can be used for tracing the biosynthetic-pathways
of secondary metabolites using labeled precursor in the culture medium.
Immobilization of cells- Tissue culture can also be used for plants preservation by
immobilization of cell further facilitating transportation and biotransformation.

History of Plant Tissue Culture

The German Botanist Guttlieb Haberlandt first proposed the importance of plant tissue
and cell culture in isolation, in 1902. He is regarded as the father of plant tissue culture.
He used tissue of Lamium Puroureum and Eichhornia crassipes, the epidemis of
Ornithooalium and epidermal hairs of Pulmonaria Mollissima. He grew them on a
particular salt solution with sucrose and observed obvious growth in the cells. The cells
remained alive for up to 1 month. They grew in size, changed shape; thickening of cell
walls occurred and starch appeared in the chloroplasts, which initially lacked it.
However, none of the cells divided. The failure was that he was handling highly
differentiated cells and the present day growth hormones, necessary for inducing division
in mature cells, were not available to him. Hanning (1940) had initiated a new line of
investigation, which later developed into an important applied area of in-vitro techniques.
Hanning excised nearly mature embryos of some plants like Raphanus Sativus and
successfully grew them to maturity on mineral salts and sugar solution. Van Overbeck
(1941) and co-workers demonstrated for the first time the stimulatory effect of coconut
milk, which was similar to embryo sac fluid, on embryo development and callus
formation in Datura. This proved a turning point in the field of embryo culture, for it
enabled the culture of young embryos which failed to grow on a mixture of mineral salts,
vitamins, amino acids and sugar. Subsequent detailed work by Raghavan and Torrey
(1963), Norstog (1965) and others led to the development of Synthetic media for the
culture of younger embryos. Laibach (1925, 1929) demonstrated the practical applicafion
of embryo culture in the field of plant breeding. He isolated embryos from nonviable
seeds of a particular plant and reared them to maturity on a nutrient medium. In 1922,
working independently Robbins (USA) and kotte (Germany) reported some success with
growing isolated root tips. White made the first successful report of continuously
growing tomato root tips in 1934. During 1939 - 1950 extensive work on root culture was
undertaken by Street to understand the role of vitamins implant growth and shoot-root
relationship. Gautheret (1934), White (1939) and Nobecourt successfully cultured cells of
Salix, Nicotiana-Hybrid and carrot on synthetic media. They, for the first time,
demonstrated that growth regulators and vitamins if added to media enhanced the growth
forming mess of cells called callus. Skoog (1944), Tsui (1951), and Miller (1955)
demonstrated the induction of divisions in isolated, mature and differentiated cells by
using synthetic as well as natural compounds. Muir (1953) developed a technique of
growing single cells into liquid medium in case of Tenetes Erecta and Nicotiana
Tabacum. Vasil and Hildeprandt (1965) raised whole plants starting from single cells of
tobacco. Skoog and Miller (1957) showed that changing the relative concentrations of the
two substances in the medium could regulate the organ differentiation. The first reports of
some embryo formation from Carrot tissue appeared in 1958-59 by Reinert (Germany)
and Steward (USA). Ball (1946) successfully raised whole plants of Lupinus and
Tropaeolum by culturing shoot tips. Morel and Martin (1952), for the first time,
recovered virus-free Dahlia plants from infected individuals by culturing their shoots.
Murashige (USA) used this technique to muUiply plants in large number for several
species ranging from ferns to foliage, flower and fruit plants. Guha and Maheshwari
(1966) demonstrated the possibility of raising large numbers of plantlets from pollen
grains of Dhatura. In 1972 Carlson and others produced the first somatic hybrid between
two plants by fiising their protoplasts.

Basic Requirement for Plant Tissue Culture

For tissue culture technique a tissue culture laboratory should have the following general
basic facilities:
1. Equipment and apparatus
2. Washing and storage facilities
3. Media preparation room
4. Sterilization room
5. Aseptic chamber for culture
6. Culture rooms or incubators fully equipped with temperature, light and humidity
control devices
7. Observation or recording area well equipped with computer for data processing
 Equipment and apparatus
Culture vessels and glassware- Many different kind of vessels may be used for wing
cultures. Callus culture can be grown successfully in large test tubes (25×150mm) or
wide mouth conical flasks. In addition to the culture vessels, glassware such as graduated
pipettes, measuring cylinders, beaker, filters, funnel, and petri dishes are also required for
making preparations. All the glasswares should be of pyrex or corning.
Equipment- scissors, scalpels and forceps for explants preparation from excised plant
parts and for their transfer.
• 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,
• 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 air flow with uv light fitting for aseptic transfer of
explants to the medium and for subculturing
• a BOD incubator for maintaining constant temperature to facilitate the culture of
callus and its subsequent maintenance
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.
Sufficient space is required for placing hot air oven, washing machine, pipette washers
and the plastic bucket or steel tray for soaking or drainage of the detergent bath or extra
water. For the storage of dried glassware separate dust proof cupboards or cabinet should
be provided. It is mandatory to maintain cleanliness in the area of washing, drying and
storage.
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 preparaed media and
stock solutions.
Sterilization room
For the sterilization of culture media, a good quality ISI marks 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.
 Aseptic chamber for culture

For the transfer of culture into sterilized media, contaminant free environment is
mandatory. The simplest type of transfer area requires an ordinary type of small wooden
hood, having a glass or plastic door either sliding or hinged fitted with uv tube. This
aseptic can be conveniently placed in a quiet corner of the laboratory.
Modern laboratory have laminar air flow cabinet having vertical or horizontal airflow,
arrange over the working surface to make it free from dust particle/micro contaminants.

Incubation room or incubator

Environmental factors affect the growth and differentiation of cultured tissues. A typical
incubation chamber or area should have both light and temperature controlled devices
managed for 24 h period.
AC or room heaters are required to maintain the temperature at 25±20c.
Light should be adjusted in the terms of photo period duration.
Humidity should be in the range of 20-90%.
Shelves should be designed in such a way so that the culture vessels can be placed in the
shelf or trays in such a ways that there should not be any hindrance in the light,
temperature and humidity maintenance. A label should be stick on the each tray and rack
to ensure identity and for maintaining the data of experiment. Label should having the
full detail about date of inoculation, name of explants, medium and any other special
information.
These days BOD incubators with all the requisite environmental condition maintenance
are available in the market.
They occupy less space and manageable with small generator or automatic inverter in the
case of electricity failure to maintain the necessary light and temperature conditions.
BOD incubators required to maintain the culture conditions should have the following
characteristics:
• Temperature range, 2-400c
• Temperature control±0.50c
• Automatic digital temperature recorder
• 24-h temperature and light programming
• Adjustable fluorescent lighting up to 10,000lux
• Relative humidity range 20-98%
• Relative humidity control±3%
• Uniform forced air circulation
• Shaker
• Capacity up to 0.7m3of 0.5m2shelf space
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. Separate dust free space should be
 marked for microscopic work. All the recorded data should be fed into the
computer.
General procedures for plant tissue culture –
• Sterilization of glassware tools/vessels
• Preparation and sterilization of explants
• Production of callus from explants
• Proliferation of cultured callus
• Sub culturing of callus
• Suspension culture
Sterilization of glassware tools/vessels- All the glassware should be of Pyrex or
corning. All the glassware should be kept overnight dipped in sodium dichromate-
sulphuric acid solution. 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. For drying the glassware, it is placed in hot air oven at
high temperature about 1200c for ½- 1 h.
In the case of plastic lab ware, washing should be carried out with a mild nonabrasive
detergent followed by washing under tap water or the plastic ware 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. Washed and dried
glassware or plastic ware should be stored in dust proof cupboards.
To prevent reinfection following sterilization, empty containers are wrapped with
aluminium foil. Stainless steel, metal tools (knives, scalpels, forceps, etc) are also
wrapped with aluminium foil and pads o cotton wool are stuffed into the opening of the
pipettes, which are either also wrapped in aluminium or placed in an aluminium or
stainless steel box. The period of sterilization usually ranges between 1 and 4 hour.
Preparation of Explant-
Explants 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. 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.
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 portion 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.
Surface sterilization of explant-
For the surface sterilization of the explant, chromic acid, mercuric chloride(0.11%),
calcium hypochlorite(1-2%) and alcohol(70%) are used. Usually the tissue is immersed in
the solution of sterilizing agent for 10 s to 15 min, and 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.
The explants are sterilized by exposing to aqueous sterilized solution of different
concentration.
Different types of surface sterilizing agent are-

Name of chemical Concentration% Exposure(min)

Bromine water 1-2 2-10
Benzalkonium chloride 0.01-0.1 5-20
Sodium hypochlorite 0.5-51 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

In the case of leaf or green fresh stem the explant needs pretreatment with wetting agent
(70-90% ethyl alcohol, Tween20), 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)
Procedure to be followed for respective explants is as follows:
Seeds-
• Dip the seeds into absolute ethyl alcohol for 10s and rinse with purified water.
• Expose seeds for 20-30 min to 10% w/v aqueous calcium hypochlorite or for 5
min in a 1% solution of bromine water.
• Wash the treated seeds with sterile water followed by germination on damp sterile
filter paper.
Fruits-
• Rinse the fruit with absolute alcohol.
• Submerge into 2% (w/v) solution sodium hypochlorite for 10 min.
• Washing repeated with sterile water and remove seeds of interior tissue.
Stem-
• Clean the explants with running tap water followed by rinsing with pure alcohol.
• Submerge into 2% (w/v) solution sodium hypochlorite for 15-30 min.
• Wash three times with sterile water.
Leaves-
Clean the leaf explant with purified water to make it free from dirt and rub the surface
with absolute ethyl alcohol. Dip the explants in 0.1%(w/v) mercuric chloride solution,
wash with sterile water to make it free from chloride and then dry the surface with sterile
tissue paper.
Production of callus from explants-
The sterilized explant is transferred aseptically onto defined medium contained in flasks.
The flasks are transferred to BOD incubator for maintenance of culture. The temperature
is adjusted to 25±20c. Some amount of light is necessary for callus (undifferentiated
amorphous cell mass) production. Usually sufficient amount of callus is produced within
3-8 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 hormone, which supports growth. The
medium used for production of more amount of callus is called proliferation medium.
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 subculturing can be done.