Assignment of Plant Tissue Culture

Topic:

Introduction to Plant Tissue Culture.

BS Botany:

7th Semester

Submitted to:

Sir Waheed Mumtaz

Submitted by:

Farrah Sajid(002)

Shehla Gul(003)

Hira Hassan(004)

Umer Yasin (005)

Rabia Aqeel (006)

CONTENT:

Introduction of plant tissue culture

History of plant tissue culture

Types of plant tissue culture

Applications of plant tissue culture

Essentials of tissue culture laboratory

Equipments and glass wares

Culture media

Media preparation

Aseptic techniques

Lab-safety
 Plant Tissue Culture

Plant tissue culture has a great significance in plant biotechnology specially in the crop
improvement programmes. The term tissue culture may be defined as the process of in-vitro culture
of explants (pieces of living differentiated tissues) in nutrient medium under aseptic conditions.
However, in general, the tissue culture includes the term tissue culture as well as cell culture, organ
culture and suspension culture also.

Plant tissue culture is fundamental to most aspects of biotechnology of plants. It is evident now
that plant biotechnology is one of the most beneficial of all the sciences. The products of plant
biotechnology are being transferred rapidly from laboratories to the fields.

History:
Although tissue culture has been around since the beginning of the 18th century, plant tissue
culture only began developing in 1898. Gottlieb Haberlandt, a German Botanist, made the first
attempt to use the in vitro method when grow plant tissues. The cells he used were varied, palisade
tissues coming from:

 the leaves
 the piths
 the epidermis and epidermal hairs

This initial experiment was fruitful for several months. However, the cells did not proliferate
further.
Over the years, many experiments used similar parameters. In these experiments, the cells survived
for a longer time, but they also failed to proliferate.

The first root tips were cultured in 1922, and, by making use of subculturing, maintained their
cultured roots for 20 weeks.

In the 1930s, it was recognized that B- vitamins and auxin (IAA) were key components in growing
root cultures using the tissue culture method.

Over the following century, many similar experiments were conducted and researchers began
identifying the most important parameters that should guide our tissue culture process today.

Whether you are in the growing industry for profit or for pleasure, the tissue culture method could
be the best thing for your buds.

Types of Tissue Culture:

Plant tissue culture have following types.

Type # 1. Seed Culture:

  Seeds may be cultured in vitro to generate seedlings or plants. It is the best method for
raising the sterile seedling. The seed culture is done to get the different kinds of explants
from aseptically grown plants which help in better maintenance of aseptic tissue.

Type # 2. Embryo Culture:

 Embryo culture is the sterile isolation and growth of an immature or mature embryo in vitro
with the goal of obtaining a viable plant. In some plants seed dormancy may be due to
chemical inhibitors or mechanical resistance, structures covering the embryo. Excision of
embryos and culturing them in nutrient media help in developing viable seedlings.
 Embryo developed from wide hybridization between two different species may not mature
fully due to embryo-endosperm incompatibility. So, the isolation and culture of hybrid
embryos prior to abortion help in overcoming the post-zygotic barrier and production of
interspecific or inter-generic hybrids.

Type # 3. Meristem Culture:

 The apical meristem of shoots of angiosperms and gymnosperms can be cultured to get the
disease free plants. Meristem tips, between 0.2-0.5 mm, most frequently produce virus-free
plants and this method is referred to as meristem-tip culture.

 This method is more successful in case of herbaceous plants than woody plants. In case of
woody plants, the success is obtained when the explant is taken after the dormancy period
is over. After the shoot tip proliferation, the rooting is done and then the rooted plantlet is
potted.

Type # 4. Bud Culture:

 Buds contain quiescent or active meristems in the leaf axils, which are capable of growing
into a shoot. Single node culture, where each node of the stem is cut and allowed to grow
on a nutrient media to develop the shoot tip from the axil which ultimately develops into
new plantlet. In axillary bud method, where the axillary buds are isolated from the leaf
axils and develop into shoot tip under little high cytokinin concentration.

Type # 5. Callus Culture:

 Callus is basically more or less un-organised dedifferentiated mass of cells arising from
any kind of explant under in vitro cultural conditions. The cells in callus are
parenchymatous in nature, but may or may not be homogenous mass of cells. They are
 meristematic tissue, under special circumstances they may be again organised into shoot
primordia or may develop into somatic embryos.

 The callus tissue from different plant species may be different in structure and growth habit.
The callus growth is also dependent on factors like the type of explant and the growth
conditions. After callus induction it can be sub-cultured regularly with appropriate new
medium for growth and maintenance.

Type # 6. Cell Suspension Culture:

 The growing of individual cells that have been obtained from any kind of explant tissue or
callus referred to as cell suspension culture. These are initiated by transferring pieces of
tissue explant/callus into liquid medium (without agar) and then placed them on a gyratory
shaker to provide both aeration and dispersion of cells. Like callus culture, the cells are
also sub-cultured into new medium.

 Cell suspension cultures may be done in batch culture or continuous culture system. In the
later system, the culture is continuously supplied with nutrients by the inflow of fresh
medium with subsequent draining out of used medium but the culture volume is constant.
This culture method is mainly used for the synthesis of specific metabolite or for biomass
production.

Type # 7. Anther Culture:

 An important aspect of plant tissue culture is the haploid production by another culture or
pollen culture which was first established by Guha and Maheswari (1964, 1966) in Datura.

 During the last few decades, much progress has been made in different crops like rice,
wheat, maize, mustard, pepper and others. The anthers bearing the uni-nucleate
microspores are selected and allowed to grow in medium to produce callus from the pollen
mass.

 Then the triggering of these androgenic calli is directed to produce the embryos and haploid
plants are developed from these androgenic embryos. The anther culture can be done with
the isolated anthers on solid medium where anther wall will break open and the androgenic
calli will be formed from the pollen.

 In pollen culture, microspores of uni-nucleate stage are collected in liquid media and can
be grown in suspension culture. In suspension, the uni-nucleate pollens may give rise to
 calli mass or the globular mass from which the plants can be raised either through
embryogenic or organogenic pathway.

Type # 8. Protoplast Culture:

 It is the culture of plant protoplasts i.e., culture of cells devoid of cell wall. Isolated
protoplasts are usually cultured in either liquid or semisolid agar media plates. Protoplasts
are isolated from soft parenchymatous tissue by enzymatic method and then viable proto-
plasts are purified and cultured.

 The protoplast culture is aimed mainly to develop genetically transformed plant where the
transgenic is put successfully within the plant protoplast and the transgenic plant is
regenerated from that transformed protoplast. Another aspect of protoplast culture is
somatic hybridization of two plant species through protoplast fusion.

Major Steps of Plant Tissue Culture:

Initiation Phase (Stage 1)

The initiation phase is the first phase of tissue culture. Here, the tissue of interest is obtained and
introduced and sterilized in order to prevent any microorganism from negatively affecting the
process. It is during this stage that the tissue is initiated in to culture.

Multiplication Phase (Stage 2)

The multiplication phase is the second step of tissue culture where the in vitro plant material is re-
divided and then introduced in to the medium. Here, the medium is composed of appropriate
components for growth including regulators and nutrients. These are responsible for the
proliferation of the tissue and the production of multiple shoots.

* This step is often repeated several times in order to obtain the desired number of plants

Root formation (Stage 3)

It is at this phase that roots are formed. Here, hormones are required in order to induce rooting,
and consequently complete plantlets.
 Applications of plant tissue culture:
1. The use of plant cells to generate useful products and or services constitutes plant biotechnology.

2. In plant biotechnology, the useful product is a plantlet. The plantlets are used for various
purposes.

3. All the cells in callus or suspension culture are derived from a single explant by mitotic division.

4. Therefore, all plantlets regenerated from a callus/suspension culture generally have the same
genotype and constitute a clone. These plantlets are used for rapid clonal propagation.

5. Genetic variation present among plant cells of a culture is called soma clonal variation.

6. A gene that is transferred into an organism by genetic engineering is known as transgene.

7. An organism that contains and expresses a transgene is s, called transgenic organism.

8. The transgenes can be introduced into individual plant cells. The plantlets can be regenerated
from these cells.

9. These plantlets give rise to the highly valuable transgenic plants.

10. Mutagens are added to single cell liquid cultures for induction of mutations.
11. Tolerance to stress like pollutants, toxins, salts, drought, flooding, etc. can also be obtained by
providing them in culture medium in increasing dosage. The surviving healthy cells are taken to
solid medium for raising resistant plants.

12. Weedicides are added to culture initially in very small concentrations.

13. Dosage is increased in subsequent cultures till the desired level of weedicide resistance is
obtained.

14. The resistant cells are then regenerated to form plantlets and plants.

15. Both apical and axillary meristems are free from virus even if the whole plant is infected by it.
Virus free plantlets are obtained by using meristem culture to grow new plants.

16. Embryos which normally do not survive inside seeds can be grown in tissue culture to form
new plants. It is useful in interspecific hybridization.

Advantages of Tissue Culture:

There are several advantages to using the tissue culture process. We already mentioned its
effectiveness in helping developing countries to increase food production, but what are some other
advantages that may be relevant to you?

 The new plantlets can be grown in a short amount of time.

 Only a small amount of initial plant tissue is required.
 The new plantlets and plants are more likely to be free of viruses and diseases.
 The process is not dependant on the seasons and can be done throughout the year.
 You need only a relatively small space to perform the process (ten times the plants in one-
tenth of the space).
 On a larger scale, the tissue culture process helps to supply the consumer market with new
subspecies and variety.
 People looking to cultivate challenging plants such as specific breeds of orchid find more
success with the tissue culture process than traditional soil.

DISADVANTAGES OF TISSUE CULTURE:

 Tissue Culture can require more labor and cost more money.
 There is a chance that the propagated plants will be less resilient to diseases due to the type
of environment they are grown in.
 It is imperative that, before being cultured, the material is screened; failure to pick up any
abnormalities could lead to the new plants being infected.
 While the success rate is high if the correct procedures are followed, success with the tissue
culture is not a guarantee. There is still a chance that the process triggers a secondary
 metabolic chemical reaction, and the new explants or cells' growth gets stunted, or even
die off.

Tissue Culture Laboratory:

An ideal tissue culture laboratory should have at least two big rooms and a small room. One big
room is for general laboratory work such as preparation of media, autoclaving, distillation of water
etc. The other big room is for keeping cultures under controlled light, temperature and humidity.
The small room is for aseptic work and for keeping autoclaved articles.

General Laboratory:
The general laboratory for tissue culture should be provided with the following articles and
arrangements:

A Washing Area:
This is very important for a tissue culture laboratory. It should be provided with a large sink,
running hot and cold tap water, brushes of various sizes, detergent and a bucket of single distilled
water for a final rinse of the washed glass goods.

A number of plastic buckets are required for soaking the glass goods to be washed. Another
separate bucket with lid is also required for disposing off the used or infected media before
cleaning. Only this bucket should be kept outside the room or cleaning area and should be cleaned
twice-in a week.

Hot Air Oven:

It is necessary for drying the washed glass goods. For this purpose, a number of enameled trays of
different sizes are required for keeping wet glass goods inside the oven.

Refrigerator:
It is essential for storing various thermo- labile chemicals like vitamins, hormones, amino acids,
casein-hydrolysate, yeast extract, coconut milk, etc. Stock solutions of salts are also kept to prevent
contamination.

Distillation Plant:
A single distillation and a double distillation water plant are indispensible. Two big plastic
containers are required for storing the distilled water.
Weighing Balance:
Three types of weighing balances viz. pan balance; chemical balance and electric balance are
required for weighing chemicals, sugars, agar- agar and others.

pH Meter:
It is necessary for the measurement and adjustment of pH of the nutrient medium.

Vacuum Pump:
It is required for filtering liquid media, sugar solution etc. through filter apparatus using air suction.

Autoclave:
It is very important for sterilization of nutrient media, glass goods, instruments, etc.
Working Tables:
These are necessary for preparation of medium.

Heater:
It is needed for heating or warming the medium to dissolve agar or to melt the agarified medium.

Microscope:
Simple, compound, inverted binocular dissection microscopes are essential for various purposes.
Some of the microscopes should be fitted with a camera for taking photomicrograph.

Microtome:
It is needed for sectioning the cultured tissue.

Wooden Rocks:
These are required for keeping the various chemicals.

Laboratory for Aseptic Inoculation:

This room should be without any window or ventilator in order to make it dust-free. The room
should be provided with double doors. The doors should have a automatic door closer. Inside floor
should be fitted with a rubber mat to facilitate cleaning. For entering into the room, shoes should
be left outside.

For aseptic work, a large wooden chamber (Ca, 4′ x 4′ x 7′) is made for short term work. Upper
half of the side walls of the chamber is made of large glass sheets. The chamber should also have
double doors provided with a door closer.

The chamber is provided with two UV (one small, one big) sterilizing lamps and a fluorescent
lamp. The switches to operate them are present outside the chamber so that the lamps can be safely
switched on and off. Inside the chamber the working table and shelves are made of thick glass
sheets.

For simple routine work such as aseptic seed germination, harvesting of cultured tissue from the
aseptic stock for cytological work or for microtome preparations, a small inoculating hood may be
used. This can be placed on a small table at the convenient corner of the room. The figure of an
ideal chamber is given here.
Laminar air flow cabinet is the most suitable, convenient and reliable instrument for aseptic work.
It allows one to work for a longer period which is not possible inside the inoculation chamber.
Long hours of work inside the inoculation chamber may also cause suffocation and needs the
interruption of work. One can work openly and easily for a longer period on the table of laminar
air flow.

Laminar air flow has a number of small blower motors to blow air which passes through a number
of HEPA (high efficiency particulate air) filters. Such filters remove particles larger than 0.3 µm.
The ultraclean air which is free from fungal and bacterial contaminants, flows at the velocity of
about 27 ±3m/minute through the working area.

All contaminants are blown away by the ultraclean air and thereby an aseptic environment is
maintained over the working area. Before starting work, laminar air flow is put on for 10-15
minutes. The flow of air does not put out the flame of a spirit lamp. Therefore, a spirit lamp can
be used conveniently during the work.

Culture Room:
The culture room means the room for keeping or incubating the culture under controlled
temperature, light and humidity. The culture room is also fitted with double doors in order to make
it dust free and to maintain a constant room temperature. One should enter the culture room
keeping the shoes outside the door. To maintain the temperature around 25 ±2 ° C in-sides the
culture room, air coolers are used.
This room is also provided with specially designed shelves (Fig. 1.7) to keep culture vessels. The
shelves are made of glass or plywood. Flask, bottles, jars; petriplates can be placed directly on the
shelves. Culture tubes can be kept on a support such as empty paper cover of fluorescent lamps.
Cultures can be grown in light or in dark.

For light arrangement, each culture rack is provided with fluorescent lamps which are photo
periodically controlled by an automatic timer. Racks covered with black curtains are suitable for
dark incubation of culture. A thermometer and a hygrometer are fixed on the wall at the safety
corner of the room to check temperature and relative humidity respectively.

The relative humidity of the culture room is maintained above 50%. Some small shelves are placed
in the culture room for temporarily keeping the autoclaved articles and the culture vials containing
the medium. The culture room should also have a shaker for suspension culture or single cell
culture in moving liquid medium. The speed of revolution of the shaker can be controlled. The
shaker is also provided with light. The platform of the shaker is fitted with clips for holding conical
flasks (150 ml to 200 ml).

Glass wares

Glassware is found in abundance in laboratories and comes in all shapes and sizes. Though it has
become preferable in recent years to substitute glass vessels for cheaper, more durable and less
fragile plastic materials, some substances and experiments or applications still require the use of
glassware.
The reasons for these are manifold. Firstly, glass is relatively inert, meaning it will not react with
the chemicals or substances placed inside and thereby upset or skew the results. It is also
transparent, allowing for easy monitoring, and heat-resistant, allowing for high temperatures.
Furthermore, it is easy to shape and mould into any form required.

The Types of Glassware:

There is a vast variety of different glass apparatuses in a laboratory, and they can be manufactured
from various types of glass depending on the purpose. For example, quartz glass is resistant to
high temperatures and transparent in specific areas of the electromagnetic spectrum. Heavy-wall
glass is specifically strengthened to be used in pressurised experiments, while amber glass is
darkened to block out UV and infrared radiation, thereby making it ideal for storing fluids.

Here are some of the different types of glass instruments used in laboratories:
 Bulb and graduated pipettes. These are used to transport specific amounts of fluids from
one place to another.
 Burettes. These are used to dispense exact quantities of liquid into another vessel.
 Beakers. Simple containers used to hold samples and reagents.
 Volumetric flasks. Similar to beakers, these are used to hold samples, but usually come in
a conical or spherical shape with a tapering neck.
 Condensers. Specifically used to cool heated liquid or gas.
 Retorts. These are used for distillation purposes.
 Funnels. The tapered neck of a funnel allows easy pouring of a liquid into a narrow orifice.
 Petri dishes. Shallow dishes used to culture living cells.
 Graduated Cylinders. Similar to beakers, these cylindrical vessels have volumetric
markings to allow for monitoring of volume.
 Vials. Small bottles used to store samples or reagents.
 Slides. Used to hold items under a microscope for inspection and study.
 Stirring Rods. Used to mix solvents and samples together.
 Desiccators. A container designed to absorb moisture from a substance.
 Drying pistols. Similar to a desiccator, the pistol is a more direct method of removing
moisture from a sample.
With such an extensive range of glassware in the workplace and maximum precision required in
all experiments, it is imperative that the equipment is kept in top-quality condition. Though glass
is resistant to high temperatures and most chemicals (barring a handful), prolonged use over a
protracted period of time will inevitably lead to degradation.

Culture media:

Culture media are largely responsible for the in vitro growth and morphogenesis of plant tissues.

The success of the plant tissue culture depends on the choice of the nutrient medium. In fact, the
cells of most plant cells can be grown in culture media.

Basically, the plant tissue culture media should contain the same nutrients as required by the whole
plant. It may be noted that plants in nature can synthesize their own food material. However, plants
growing in vitro are mainly heterotrophic i.e. they cannot synthesize their own food.

Composition of Media:
The composition of the culture media is primarily dependent on two parameters:

1. The particular species of the plant.

2. The type of material used for culture i.e. cells, tissues, organs, protoplasts.

Thus, the composition of a medium is formulated considering the specific requirements of a given
culture system. The media used may be solid (solid medium) or liquid (liquid medium) in nature.
The selection of solid or liquid medium is dependent on the better response of a culture.
Important constituents of a culture medium are:
(i) Organic supplements:
 (a) Vitamins like thiamine (B1), Pyridoxin (B6), Nicotinic Acid (B3), etc.
 (b) Antibiotics like Streptomycin, Kanamycin;

 (c) Amino Acids like Arginine, Asparagine.

(ii) Inorganic Nutrients:

 Micronutrients as Iron (Fe), Manganese (Mn), Zinc (Zn), Molybdenum (Mo), Copper
(Cu), Boron (B).

 Macronutrients include six major elements as Nitrogen (N), Sulphur (S), Phosphorus (P),
Potassium (K), Calcium (Ca), Magnesium (Mg).

(iii) Carbon and Energy Source:

 Most preferred carbon source is Sucrose. Others include lactose, maltose, galactose,
raffinose, cellobiose, etc.

(iv) Growth Hormones:

 a. Auxins-mainly for inducing cell division.

 b. Cytokinins-mainly for modifying apical dominance and shoot differentiation.

 c. Abscisic Acid (ABA)-Used occasionally.

 d. Gibberellins-Used occasionally.

Gelling Agents:
 These are added to media to make them semisolid or solid. Agar, Gelatin, Alginate etc.
are common solidifying or gelling agents.

Other Organic Extracts:

 Sometimes culture media are supplemented with some organic extracts also like coconut
milk, orange juice, tomato juice, potato extract, etc.

Types of media:
There are a number of culture mediums known to date such as MS medium, B5 medium, LS
medium, White’s medium, etc.

Murashige and Skoog (MS) medium:

This medium was invented by two scientists named Toshio Murashige and Folke K. Skoog
in 1962 while the two scientists were working on the discovery of plant growth regulators.
It is the most commonly used medium in the tissue culture lab.

Sometimes you may observe some numbers behind the MS, which indicate the
concentration of sucrose in the medium. For example, MS0 indicates sucrose absence and
MS10 indicated the presence of 10g/l sucrose in the medium. The formulation is a blend
of nutrients like inorganic salts, vitamins, and amino acids.

Purpose: This medium is used to induce organogenesis, callus culture, micropropagation,

and cell suspension.

1. Linsmaier and Skoog (LS) medium:

This medium was developed by Linsmaier and Skoog in 1965. It was first used to optimize
the organic supplements of the tobacco culture. The medium has a similar component as
Murashige and Skoog with a twist of Linsmaier and Skoog vitamins.

He found that the increased concentration of thiamine hypochlorite (0.4mg/l rather than
0.1mg/l) compensated for the absence of vitamins except for inositol. Inositol is an
enzymatic cofactor in glycolysis and TCA cycle and it is also involved in primary and
secondary metabolism of the plants.

Purpose: It is used for the purpose of organogenesis, callus culture, cell suspension, and
micropropagation.

2. Gamborg (B5) medium:

This medium was developed by O. L. Gamborg in 1968. He used the media for the callus
and cell suspension culture of Glycine max belonging to the family of Fabaceae. This
medium is a blend of nutrients like inorganic salts, vitamins, and carbohydrates.

The medium has a higher concentration of nitrate and potassium and a lower concentration
of ammonia. Potassium nitrate is useful in inducing the soybean root callus formation and
ammonium sulfate plays an essential role in cell growth.

Purpose: It is used for the purpose of protoplast culture.

3. Nitsch and Nitsch (NN) medium:

 The medium was developed by J. P. Nitsch in 1969 for the establishment of the in vitro
anther culture of Nicotiana, from family Solanaceae. It contains a high concentration of
thiamine, biotin, and folic acid that supports anther callus.

Purpose: To establish the in vitro anther culture.

4. White’s Medium:

The medium was developed by P. R. White in 1963 for the establishment of the root culture
of tomato. This was the earliest plant tissue culture media developed for root culture. It has
a lower salt concentration and a higher concentration of MgSO4. The concentration of
nitrate is 19% lesser than the MS media.

Purpose: White’s medium can be used for the purpose of shoot culture and callus culture.
It is suitable for culture Musa and Daucus species.

Media selection:
It is a very crucial step in tissue culture but there isn't one step to formulate a suitable media for
the establishment of the culture. However, you can start working with three media having different
salt concentrations, such as high salt concentration, medium salt concentration, and low salt
concentration, to get the desired response from the culture.

A combination of different ratio of auxin and cytokinin can be used for shoot proliferation or
adventitious shoot formation. Furthermore, a range of sucrose concentration (2-6%) can be tested
to establish the culture. So, there are various opportunities to make the desired medium suitable
for your culture by simply manipulating the concentration of nutrient salt and plant growth
regulators.

A broad spectrum experiment is described by De Fosard et al to create the suitable media for your
culture, especially if the system is untested.

1. Divide the medium into four broad categories: (a) minerals, (b) auxins, (c) cytokinins, and
(d) organic nutrients (sucrose, amino acids, inositol, etc.)
2. For each group of the component, choose three concentrations: high, medium, and low.
3. Try various combinations of the four groups of the components into three different
concentrations. This will lead to an experiment with 81 treatments.
4. Denote the best of 81 treatments with four-letter code. For example, the treatment with
medium salts, low auxin, medium cytokinin, and high organic nutrients would be
represented as MLMH.
5. After reaching this stage different concentrations of auxin and cytokinin can be tested to
determine the suitable concentration of growth regulators.
 Preparation of Media:

The general methodology for a medium preparation involves preparation of stock solutions (in the
range of 10x to 10Ox concentrations) using high purity chemicals and demineralized water. The
stock solutions can be stored (in glass or plastic containers) frozen and used as and when required.
Most of the growth regulators are not soluble in water. They have to be dissolved in NaOH or
alcohol.

Dryer powders in Media Preparation:

The conventional procedure for media preparation is tedious and time consuming. Now a days,
plant tissue culture media are commercially prepared, and are available in the market as dry
powders. The requisite medium can be prepared by dissolving the powder in a glass distilled or
demineralized water. Sugar, organic supplements and agar (melted) are added, pH adjusted and
the medium diluted to a final volume (usually 1 litre).
Sterilization of Media:
The culture medium is usually sterilized in an autoclave at 121°C and 15 psi for 20 minutes.
Hormones and other heat sensitive organic compounds are filter-sterilized, and added to the
autoclaved medium.

Selection of a Suitable Medium:

In order to select a suitable medium for a particular plant culture system, it is customary to start
with a known medium (e.g. MS medium, B5 medium) and then develop a new medium with the
desired characteristics. Among the constituents of a medium, growth regulators (auxins,
cytokinins) are highly variable depending on the culture system.

In practice, 3-5 different concentrations of growth regulators in different combinations are used
and the best among them are selected. For the selection of appropriate concentrations of minerals
and organic constituents in the medium, similar approach referred above, can be employed.

Medium-utmost Important for Culture:

For tissue culture techniques, it is absolutely essential that the medium preparation and
composition are carefully followed. Any mistake in the preparation of the medium is likely to do
a great harm to the culture system as a whole.

One of the most common problems in tissue culture lab that cost us labor, money, and productivity
of our cultures is “contamination”. The other problem is that the source of this contamination is
not fixed. The microbes, invisible to naked eyes, can attack and invade our cultures through any
tools, chemicals, explants, or even our hands! And you won’t realize until they multiply in numbers
and form large colonies on the media. Now, you will ask, what can you do about it? How can you
save your lovely cultures from this alien attack!

The first requirement while performing tissue culture is a “sterile environment”. You must sterilize
your equipment, media, explants, culture area, hands, etc. However, different tools and materials
require different techniques for their sterilization.

ASEPTIC TECHNIQUES:
These are techniques or procedures that aid in preventing contamination from our cultures.
Different types of aseptic techniques include dry heat, wet heat, ultrafiltration, chemical
sterilization, antibiotics, and PPM™.

How do these techniques work?

 Dry Heat: This technique is used to sterilize equipment like glassware and metal
instruments without using extra moisture.
  Wet Heat: This technique involves autoclaving, which acts as a pressure cooker and
produces heat to sterilize equipment, paper products, glassware, and liquid substances.
 Ultrafiltration: This is the best method to sterilize the media because it doesn’t allow any
reaction between media components during the process. Membrane filtres used in this
method have micro pores in them that restrict microbes from passing the filter.
 Chemical Sterilization: Use 70% isopropanol to sterilize the instruments and working area.
However, use calcium chlorite or sodium chlorite for surface sterilization of the starting
material or explant. You can also use PPM for this purpose (The best part: it doesn’t
damage your explants).
 Antibiotics: Antibiotics are not recommended for the tissue culture process as they
negatively affect the growth of cultures.

The tissue culture is an efficient technique in the research and commercialization of secondary
metabolites, horticulture crops, and plants that are difficult to grow by conventional methods.
Some people have started utilizing and working with this technique on a smaller scale as well
because of several advantages it provides.

Lab Safety:
DO’S (NEVER MISS THESE POINTS AND CAREFULLY PERFORM YOUR
EXPERIMENT)

1. Always wear personal protective equipment (PPE): To save your cultures from getting
contaminated and yourself from harmful chemicals always wear a mask, gloves, disposable
caps to cover hair, and a clean lab coat. While working with liquid nitrogen, wear thermally
isolated gloves, eye protection, and a splash-proof apron.
2. Try to keep two PPE kits for the washing and media preparation area and the tissue culture
area of the lab. You can make it easier by keeping two different colored coats in both labs.
3. Keeping the culture area, media preparation area, and transfer area clutter-free helps to
reduce mistakes while performing a particular task. It clears your mind and you will be
able to focus on the task at hand.
4. The most common mistake done while working in the lab is preparing multiple reagents or
solutions simultaneously and not labeling them. This creates confusion and may lead to the
disposal of solutions. So, always label the solutions or reagents with content and dates of
preparation as soon as you prepare. This also helps to avoid the use of a solution after its
expiration date.
5. Work with one kind of tissue or perform one experiment at a time. Don’t try to multitask
but be arranged as much as possible. This will also reduce the chances of cross-
contamination by mislabeling and the spread of microbes from numerous opened media
bottles due to the generation of aerosols.
6. Always use 70% isopropanol or ethanol to clean the work area or surface. You can use a
clean paper towel to wipe the area. This should be done before you initiate the experiment
or in between operations.
 7. If you are working with multiple cell lines then it’s advisable to maintain separate bottles
of media for each cell line in culture/cultivation.
8. Regularly examine your cultures and commercially purchased media for any signs of gross
bacterial and fungal contamination. This also prevents the spreading of microbial
contamination (if any) invaded your cultures.
9. After you buy media or reagents always check its quality control information and ideally
examine its performance before involving it in your experiments. Checking the information
will tell you what conditions are required to store your chemicals to maintain their
performance.
10. Keep minimum cardboard packaging in all the cell culture areas.
11. Always ensure at regular intervals that all the machines installed in the media preparation
area, transfer area, and culture area are working properly.
12. Mycoplasmas are endophytic bacteria that live inside cells. So, always test explants/starting
material whenever you are going to work with any for the presence of this bacteria.

DON'TS (YOU CAN PROTECT YOUR CULTURES BY JUST AVOIDING THESE

SIMPLE THINGS)

1. The continuous use of antibiotics should be strictly avoided as this can lead to the
generation of resistant strains in the culture and may mask the underlying contamination.
The use of antibiotics is also avoided in plant tissue culture because it slows down the
growth of cultures.
2. Avoid the accumulation of any kind of waste in microbiological safety cabinets or
incubators.
3. Do not make the culture area crowded at one time. It is advisable to have only one person
at a time while working especially in the culture or transfer area.
4. Don’t bring cells from unauthenticated sources in the main culture area without completing
the quality control checks. You can also use a cell supplier that authenticates cells.
5. Don’t keep cell lines continually in cultures without returning frozen stock.
6. Avoid cultures to run out of media. So, always subculture them after 70% of the media is
utilized or after a fixed interval of days suitable for a particular culture.
7. Do not allow the media to cross the expiration date. The shelf life is only 4-6 weeks at
+4°C after adding glutamine and serum.
8. Don’t let the water baths get dirty. So, clean them regularly.
9. Don’t allow the essential equipment used in the labs to become out of calibration. This will
reduce analytical mistakes.
Refrences:
1. https://www.biologydiscussion.com/botany/tissue-culture/tissue-culture-
definition-history-and-importance/42944
2. https://www.biologydiscussion.com/essay/plant-breeding-essay/essay-on-plant-
tissue-culture-history-methods-and-application/17639
3. https://www.biologydiscussion.com/plant-tissues/culture/applications-of-plant-
tissue-culture-16-applications/15688
4. https://www.biologydiscussion.com/plant-tissues/culture/plant-tissue-culture-
laboratory-with-diagram/14561
5. https://www.plantcelltechnology.com/pct-blog/advantages-and-disadvantages-of-
plant-tissue-culture
6. https://www.biologydiscussion.com/plant-tissues/culture/plant-tissue-culture-
laboratory-with-diagram/14561
7. https://www.plantcelltechnology.com/blog/elevate-your-culture-7-essential-tissue-
culture-tools/
8. https://www.labmate-online.com/news/laboratory-products/3/breaking-
news/what-are-the-different-types-of-laboratory-glassware/32653
9. https://www.plantcelltechnology.com/pct-blog/tissue-culture-medium-types-and-
5-steps-of-selection/
10. https://www.biologydiscussion.com/plants/plant-tissue-culture/plant-tissue-
culture-media-types-constituents-preparation-and-selection/10656
11. https://www.plantcelltechnology.com/blog/aseptic-techniques-subculturing-of-
staurogyne-repens/
12. https://www.plantcelltechnology.com/blog/dos-and-donts-of-tissue-culture/