EXPERIMENT: 1- Identification and use of equipments in tissue culture Laboratory.

Introduction Plant tissue culture techniques have played a pivotal role in addressing many
questions of basic and applied fields of plant science. Initially, this technique was used for
studying the roles of hormones in cytodifferentiation and organogenesis. It is now also used for
the functional characterization of genes, elucidation of underlying regulatory mechanisms by
developing genetically modified (transgenic) plants. Due to their intensive use in various fields
of applied plant science, plant tissue culture techniques have also revolutionized the agriculture
sector in modern times. For example, selected plants tissues/cells are cultured as suspended cells
to produce plant products. Additionally, transgenic plants with enhanced agronomic traits such as
high carotenoids (golden rice) or longer shelf life (Flavr Savr tomatoes) have been produced.
Further, tissue culture methods also help in developing the somatic haploid embryos. Such
somatic haploid embryos can subsequently be used for the production of homozygous plants.
Hence, tissue culture techniques have played an important role in the progression of academic
and applied plant science.

The major applications of plant tissue culture techniques are:

 The large scale production of ornamental plants.

 Conservation of endangered plant species.
 Production of plant-derived secondary metabolites and recombinant proteins on a largescale
in liquid culture of plant cells in bioreactors.
 Production of novel hybrids by either fusing protoplasts of distantly-related species or
embryo-rescue technique.
 Molecular, pharmacological and biochemical investigations of different aspects of plant
growth and development such as in vitro flowering.
 For the induction of polyploidy, by treating plants with of antimitotic agents such as
colchicine.
 To produce virus free plants from virus-infected stock, such as potatoes by culturing
meristem/tip culture.
 Production of genetically modified crops with improved agronomic traits.

Any laboratory, in which tissue culture techniques are performed, regardless of the specific
purpose, must contain a number of basic facilities. These usually include the following:

 A general washing area

 A media preparation, sterilization, and storage area
 An aseptic transfer area
 Environmentally controlled incubators or culture rooms
 An observation/data collection area
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.

Aseptic or 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.

Environmentally controlled incubators or culture rooms: 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±20 c. 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.
The applications and principle of each of the tools and techniques used for plant tissue culture
are briefly described hereunder with schematic diagrams.

1. 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

2. pH and pH Meter:

The hydrogen ion concentration of most solutions is extremely low. In 1909, Sorenson
introduced the term pH as a convenient way of expressing hydrogen ion concentration. pH of a
solution is strictly defined as the negative logarithm of the hydrogen ion activity. But in practice
usually hydrogen ion concentration is taken.
The pH probe measures pH as the activity of hydrogen ions surrounding a thin walled glass bulb
at its tip. The probe produces a small voltage (about 0.06 volt per pH unit) that is measured and
displayed as pH units by the meter. The meter circuit is fundamentally no more than a voltmeter
that displays measurements in pH units instead of volts.

3. Autoclave:

The purpose of aseptic technique in plant tissue cultures is the elimination of microorganisms
during experimental procedures. If sterile tissues are available, then the eradication of
microorganisms is done by using sterile instruments and culture media. Media and other
apparatus are made sterile by autoclaving them at 15 lbs/square inch (121°C) for 15 minutes. The
use of disposable sterile plastic ware reduces the need for autoclaving, however, augments the
total cost of the experiment. Alternative sterilization techniques such as filter sterilization (FS)
must be used for heat-labile substances like plant growth regulators (e.g. cytokinins, auxins etc.)
and antibiotics (such as kanamycin, ampicillin etc.)

3. Micropipettes:

Micropipette is used (i) to take micro quantity of any liquids accurately and (ii) to avoid the risk
of sucking toxic/poisonous chemicals in the mouth.

There are two types of micropipettes:

(a) Fixed volume: The volume of micropipette is fixed. E.g. 10 μl.

(b) Variable volume: Three categories micropipettes are commonly used in the plant tissue
culture and biotechnology laboratory with the following measuring ranges: (i) 0.5 to 10 µ1 (ii)
10 to 100 µ l (iii) 100 to 1000 µ l.
An essential component of the micropipettes is a tip that fits on the end of pipettes. The tip holds
the liquid that is being measured. There are two kinds of tips available in the markets: Sterilized,
disposable tips and Autoclavable tips.

4. Weighing Balances: It is used to weigh the exact quantity of any substances or chemicals
accurately and speedily. Most modern balances have single pan digital balances. It is preferable
to install an electronic digital balance with sensitivity of 0.001 g. One must read the instruction
given in the manual to become familiar with the balance. Chemicals are weighed by placing
them on a piece of weighing paper, in a weigh boat, or in a beaker. After placing the weighing
paper, use tare that resets the balance to zero so that one may read the desired weight directly.

5. Centrifuge: It is used to separate the cells, macromolecules such as DNA, RNA and proteins
or sub cellular organelles including mitochondria, ribosome, and nuclei by homogenizing the
cells. The centrifuge may be used either preoperatively for the isolation of pure components or
analytically to determine sedimentation rate. The centrifuges vary in the sample size they will
hold and in the centrifugal force they can generate. Force (g) is determined by the following
formula. Force (g) = 11.18 r (rpm / 1000)2 Where, r = radius of the centrifuge rpm = revolutions
per minute.
Types of centrifuges: There are two types of centrifuges in general.

(a) Low-speed centrifuges: The low-speed centrifuge has a fixed angle rotor and holds a
maximum tube size of 15 ml. The low- speed centrifuge is capable of 4000 rpm and is not
adjustable.

(b) Micro centrifuges: micro centrifuge has a rotor that will hold 1.5 ml micro centrifuge tubes.
The speed may be adjusted from 1000 rpm to a maximum speed of 20,00 rpm.

6. Hot air oven: It is a cabinet which provides a high temperature in a varied range. It is used for
general drying of glasswares at 75-80 OC and also for dry air sterilization at a temperature equal
to 125 OC for 4 – 5 hours. The glasswares are arranged in the oven such that proper circulation
of air is possible. It should not be overloaded with glasswares or plastic wares and should be
cleaned at regular intervals.

7. Laminar Air flow Cabinet: It is a cabinet or chamber having a constant blow of sterile air
towards outer direction, which provides sterile air towards outer direction, which provides sterile
conditions for the inoculation of material in culture vessels. It provides platform for aseptic work
during inoculation of explants, separation and transfer of plantlets from one glass vessel to
another.
Principle: A motor sucks air from the lower side of the cabinet and blows it in the working area
of the bench towards the outer side. Prior to blowing, the air is passed through two different two
filters, which render the air sterile.

(a) Pre-filter: It is present on lower side, which traps coarse dust particles from the incoming air.

(b) HEPA (High Efficiency Particulate Air) Filter: This filter having pore size less than 0.03
μm which removes all microorganisms (bacteria, fungi, spores etc) from the air. The sterile air is
then blown in the working area of the bench towards outer 3 m per minute, which is adequate to
direction with a velocity of equal to 27 prevent the dust particles from setting on the bench. A
bench is made up of stainless steel and contains added arrangements like a UV light, a
fluorescent light and a Bunsen burner. An ultraviolet (UV) germicidal light is kept on the roof of
the cabinet for per sterilization of the interior surface of the bench prior to work. A fluorescent
light is also kept for providing lights during work. Before starting work, it is desirable to swab
down the inside of cabinet and table top with 70 % alcohol.

Types of Laminar Airflow Cabinets: It is of two types.

(a) Horizontal Laminar Airflow Cabinets: They are preferable for plant tissue culture work,
which flow the air from back towards to the front.

(b) Vertical Laminar Airflow Cabinets: They are preferable for working with pathogens or
Agro-bacterium mediated genetic transformation which flow air from upper (top) towards the
lower (bottom) sides. If tissue culture work itself requires screening against pathogens or
isolation of toxin etc. then the pathological work are must be separated.

8. Magnetic stirrer (with hot plate): It is a stirring device used to induce variable stirring speed
for preparation of solutions, stock solutions and media with help of magnetic beads in a non-
magnetic vessel. It is also used to boil agar- agar with media before autoclaving or melting of
agarose for preparation of gel. In contains a metallic top on which the vessel containing the
solution to be mixed is placed.

It contains a speed regulator to achieve the desired speed of stirring. Magnetic stirrers with
heating arrangement (hot plate) are available in the market, which are used to heat the solution
simultaneously during stirring. Magnetic stirrer with hot plate is very beneficial as it shortens the
time required for dissolution of the chemicals having poor solubility.

9. Orbital Shaker: It is used to agitate explants for rapid multiplication in medium, suspension
culture or dissolving substances in liquid medium. Generally, suspension culture is carried in
conical flasks with a long neck. For suspension cultures, orbital shaker which having slots for
placing conical flasks of different capacities are required so that proper aeration is provided to
cells because the cells may die out due to less concentration of oxygen in the liquid medium. The
speed of the orbital shaker can be adjusted according to the requirement.

10. B.O.D. Incubator: It is used to maintain the low and high temperature during incubation of
cultures, preservation of vitamins, hormones and chemical’s stock solution at low temperature.
Simple incubators can be used to control the proper incubation temperature of the cultures. Now-
a-days, environmental shakers or orbital shaker cum incubators are available in the market in
which arrangements for providing appropriate photoperiod and temperature are made so that all
the cultures get suitable conditions for growth.

11. Maximum and Minimum Thermometer: It is a devise to record the maximum and
minimum temperature of the environment. It is an indispensable part of the plant tissue culture
laboratory since constant monitoring of the temperature of the culture room is required.
Principle: When temperature rises, mercury expands in one arm and shrinks in the other arm.
Similarly, when the temperature falls, mercury shrinks in one arm and expands in the other arm.
It has an H-shaped tube fitted on a steel plate having a marked index. The tube is filled with
mercury and alcohol. As temperature rises mercury expands and along with it the needle also
moves upwards. The position of the needle can be read from the index, which indicates the
temperature.
12. Lux Meter (Photo Meter OR Digital Light Meter): It is a devise to measure the intensity
of light. Plant cells required light in the visible range of spectrum (between wavelength 380-760
nm) for growth, which is provided by the fluorescent tube lights in the culture room. The
intensity of light required is usually around 2000-2500 lux but it varies from time to time for
various experiments. Therefore, culture room is provided with a lux meter, which records the
intensity of light. Principle: The lux meter contains a photoelectric cell and a micro ammeter.
The photoelectric cell is sensitive towards light and converts the light signals to electric current
due to which a deflection is provided in the needle of ammeter, which can be read with the help
of the calibrations provided on the scale underlying the needle.
EXPERIMENT: 2- Preparation of Solutions
Solution: It is a mixture of liquid where the minor component is solute and is dissolved in the
major component is solvent. This solute and solvent are uniformly distributed.

1. Molar solution
1. It contains one mole as (molecular weight) of solute in a solution (solvent) making it
equal to one liter.
2. Molar solution = Molecular weight in gram/liter in the solution.
3. Example:
4. I molar solution of sodium chloride (NaCl).
5.
Sodium atomic weight = 23
Chloride atomic weight = 35.5
Total molecular weight = 58.5 gram/mol
Now dissolve 58.5 grams of NaCl in distilled water and make the solution to one liter.

2. Normal solution
 The normal solution is defined as the gram equivalent weight per liter of the solution
(solvent).
o Normal solution  =  gram equivalent weight of solute/liter of the solution (solvent)
= Eq.wt/L.
 These solutions are expressed as N.
o Gram equivalent weight = Gram molecular weight/valency.
 Example of Gram equivalent weight  e.g NaCl
o NaCl gram molecular weight = 58.5 g
o Valency =1
o 58.5/1 = 58.5 gram equivalent weight.
3. Percent solution
1. This is per hundred part of the total solution.
2. There are three possibilities for a percent solution.
3. Weight/weight:
1. It is a percentage of solute in 100 grams of final solution equal to solute + solvent.
2. e.g.For the 5% solution take 5 grams of NaCl dissolved in 95 grams of water
which is around 95 mL.
4. Weight/volume:
1. 5 grams of NaCl dissolved in water and the volume is made 100 ml is called a 5%
solution of NaCl.
5. Volume/volume:
1. It is composed of two solutions. e.g. if we take 5 mL of acid and dilute it to 100
mL of water will be a 5% solution of that acid.
Preparation of stock solution: is the most basic and important steps in the plant tissue culture
technology because to prepare any basal medium the ingredients, macronutrients, micronutrients,
Iron EDTA and vitamins are common. So, in order to prepare any basal medium it is
complicated process of weighing the ingredients of micronutrients, macronutrients, vitamins,
iron EDTA each and every time and it is time consuming process. To avoid all the above
difficulties it is better to prepare stock solutions of standard concentration and use whenever
needed.

Requirements:
Chemical: Mineral salts
Glassware: Amber colour bottles, funnel, beaker, glass rod, filter paper, pipette etc.
Instrument: Autoclave

Procedure:
1. Preparation of Macronutrients stock:
Normally stock solutions of macronutrients are prepared at 20X concentration. Weigh the
ingredients as given in the table as dissolve one by one make up to volume 1000ml that is 20X
concentrations. For the preparation of 1litre of basal medium 50ml of above solution is required.
2. Preparation of Micronutrients stock:
Normally stock solutions of macronutrients are prepared at 100X concentration. Weigh the
elements as given in table and dissolve in 1000ml of distilled water that gives 100X
concentrations. To prepare 1 liter basal medium 10ml of micronutrients solution is required.
Iron-EDTA: An iron stock is prepared separately because of the problem of iron solubility. This
element requires acidic condition for solubility. Usually the iron stock is prepared in cheated
form as the Sodium salt of Ferric-Ethylene Diamine Tetra Acetic Acid (Fe-EDTA).
3. Preparation of Vitamins stock:
Weigh each vitamin as given in the table and dissolve in 1000ml of distilled water that gives
100X concentrations, 1ml is required for preparation of 1litre basal medium.
4. Preparation of Hormone Stock Solution:
Various hormones of auxins (dissolved in 70% alcohol, 1N HCl) and cytokinins (1N NaOH) are
prepared at a concentration of 1mg/ml in suitable solvent.

Table for stock solution preparation

Note:
1. All stock solution should be clear and transparent, free of dust and without precipitation.
2. Stock solutions should be stored in amber colour bottles.
3. Stock solutions are kept at 4˚C in dark room. These solutions are stored for limited period of
times.
4. After using stock solution immediately keep close and don’t expose for longer time.