BTE-308: Plant Biotechnology and

Genetic Engineering

Lecture-4
Tissue Culture: Part III
Tissue Culture Methodology

mmh.akash@bracu.ac.bd
Plant Tissue Culture Methodology
 Plant Tissue Culture
Plant tissue culture is a collection of techniques used to maintain or grow
plant cells, tissues or organs under sterile conditions on a nutrient culture
medium of known composition.

Plant tissue culture is the FOUNDATION of plant biotechnology as fully

functional plants must be grown from the genetically modified cells/tissues.

Whole plants can be regenerated in vitro using tissues, cells, or a single

cell to form whole plants by culturing them on a nutrient medium in a
sterile environment.
 Plant Tissue Culture
Elite varieties can be clonally propagated, endangered plants can be
conserved, virus-free plants can be produced by meristem culture.

Germplasm can be conserved, secondary metabolites can be produced by cell

culture.

And of course, tissue culture serves as an indispensable tool for transgenic

plant production. For nearly any transformation system, an efficient
regeneration protocol is imperative.

This can be attributed to totipotency of plant cells and manipulation of the

growth medium and hormones.
 Plant Tissue Culture
However, having an understanding of each plant species, different explants
are essential to the development of efficient regeneration systems for
different plant species.

The physiological stage of the explant plays a very important role in its
response to tissue culture.

For example, young explants generally respond better than do older ones.
 Steps for Plant Tissue Culture
1. Preparation/Sterilization of instruments

2. Preparation and Sterilization of culture medium

3. Preparation of explant

4. Inoculation of Explant

5. Incubation for growth

• Callus formation
• Shoot induction
• Root induction

6. Acclimatization of plantlets (TCPs)

7. Transfer to pots
 1. Preparation of Instruments
Apparatus required for plant tissue culture are-

Forceps, Scissors, Blade holders

Surgical blades or scalpel

Petri plates w/ two filter papers

Beaker (500mL)

Erlenmayer flask

Test tube

Micropipette and pipette tips

Bottle (1000mL)
 1. Preparation of Instruments
Apparatus other than glass- or plasticware

All the apparatus are taken in a spotlessly clean condition.

These items are then wrapped in newspaper or brown paper.

These are then kept ready for autoclaving.

 1. Preparation of Instruments
Glass- or plasticware. These apparatus are washed in a four-step process.

1. Glass and plasticwares with used cultures and media are autoclaved for
about one hour.

2. The glass- and plasticwares are then immerged in a tub full of chromic acid
for 16 hours.

3. The glass- and plasticwares are then rinsed with water to remove the
chromic acid and then immersed in a tub full of detergent water and
cleaned using hard nylon brushes

4. The glass- and plasticwares are finally immersed in another tub full of clean
water and then the detergent is rinsed off. These are then washed under
running tap water and put in an oven maintained at 60-800C for drying.

The glass and plasticwares are now READY for use!

2. Preparation of Culture Medium
 Culture Medium
Media provides optimum nutritional support for growth and morphogenesis
of in vitro grown plant tissue.

A complete plant tissue culture medium may consist of-

Nutrient medium or Basal medium

Growth regulators

And in specific cases, Gelling or solidifying agents

 Nutrient or Basal or Growth medium
The success of tissue culture lies in the composition of the growth medium,
hormones, and culture conditions such as temperature, pH, light, and
humidity.

The growth medium is a composition of essential minerals and vitamins that

are necessary for a plant’s growth and development.

It contains some macro- and micro-nutrients, vitamins and also a source of

carbon.

Today, the most widely used medium for tissue culture is Murashige and
Skoog or MS medium.

Basal medium may be supplemented with growth hormones and/or other

defined or undefined substances to obtained required regeneration or
product.
Composition of
MS medium.
 Nutrient or Basal or Growth medium
Iron is seldom added directly to the medium, it is chelated with EDTA so that it is
more stable in culture and can be absorbed by plants over a wide pH range.

If iron is not chelated with EDTA, it forms a precipitate, especially in alkaline pH.

Vitamins are necessary for the healthy growth of plant cultures.

In addition, plants require an external carbon since cultures grown in vitro rarely
photosynthesize sufficiently to support the tissues’ carbon needs. The most
commonly used carbon source is sucrose.

The pH of the medium is important since it influences the uptake of various

components of the medium as well as regulating a wide range of biochemical
reactions occurring in plant tissue cultures.

Most media are adjusted to a pH of 5.2–5.8.

 Nutrient or Basal or Growth medium
Cultures can be grown in either liquid or solid medium.

The medium is most often solidified as it provides a support system for the
explants and is easier to handle.

Solidification is done using agar derived from seaweed or agar substitutes such
as GelriteTM or PhytagelTM.
 Nutrient or Basal or Growth medium
A plethora of media formulations are available for plant tissue culture other
than MS.

Different media are suitable for culturing different types of plants.

For example-
McCown’s woody plant medium (WPM) has been widely used for tree tissue
culture.

Knudson’s medium was developed for orchid tissue culture and is also used for
fern tissue culture, etc.
 Growth Regulators
The basal medium (e.g., MS) is designed to keep plant tissues alive and
thriving.

On the other hand, plant growth regulators or hormones are needed to

manipulate the developmental program of tissues—for example, to make
callus tissue proliferate, or produce roots from shoots.

The most important growth regulators for tissue culture are auxins, cytokinins,
and gibberellins.
 Hormone Physiology in Plants
SEVEN plant hormones regulate the developmental and physiological events
in a plant.

Plant growth hormones are small molecules rather than proteins or peptides,
and in some cases they are similar to certain animal cell hormones.

Examples include-
Auxin
Cytokinin
Gibberellin
Abscisic Acid
Ethylene
Jasmonate
Brassinolide
Hormone Physiology
in Plants
Among these, THREE
phyto-hormones have
important roles in plant
tissue culture-

AUXIN

CYTOKININ

GIBBERELLIN
 Role of Different Hormones in Plants
Auxin or indole 3-acetic acid
The first plant hormone discovered

The natural functions of Auxins are-

Elongation of stem and internodes

Apical dominance

Tropism (to light or gravity)

Root initiation and lateral root development

But in tissue culture, Auxins are particularly important for the

ROOTING of shoots!
Role of Different Hormones in Plants
Auxins used in tissue culture-

Rooting of shoots
IBA (Indole-3-butyric acid)
NAA (naphthalenacetic acid)
IAA (Indole-3-acetic acid)

Callus induction and growth

2,4-D (dichloro-phenoxyacetic acid)
 Role of Different Hormones in Plants
Cytokinin
Is generally considered the second most important phyto-hormone.

The natural functions of Cytokinins are-

Cell division

Modification of apical dominance

Shoot differentiation

But in tissue culture, Cytokinins are important for-

Cell division and differentiation of adventitious shoots from callus

Shoot proliferation
Role of Different Hormones in Plants
Cytokinins used in tissue culture-

BAP (benzylamino purine)

Kinetin (furfurylamino purine)
Zeatin (naturally occurring)
Role of Different Hormones in Plants
Function of Gibberellins in tissue culture-
Elongation of shoot under in vitro condition

Inter-nodal elongation

Very rarely used hormone.

Example- GA3
Role of Different Hormones in Plants
Growth Regulators used in Tissue Culture
 Growth Regulators used in Tissue Culture
In addition to auxins and cytokinins, other hormones such as abscisic acid and
jasmonic acid have also been used in plant cell culture.

Other adjuvants (additional components that enhance growth) that have

known to have a positive effect on morphogenesis are polyamines such as
spermidine, spermine, and putrescene.

By manipulating the amount and combination of growth hormones,

regeneration of whole plants from small tissues is possible.
Brassica juncea plants produced from hypocotyl explants. Shoots are produced
when a combination of auxin and cytokinin is used.

(a) Callus from hypocotyl explants

(b) shoots from callus
(c) shoots elongating
(d) whole plantlets transferred to soil.
Preparation of Media
 Preparation of Stock Solutions
In a tissue culture medium, the quantity of a single component is often too
small which makes weighing of that component precisely very difficult.

For this reason, it is better to prepare stock solutions.

A stock solution might be either weak (10X) or very concentrated (100X).

For the preparation of stock solutions, necessary chemicals should be highly

purified (plant tissue culture tested grade or at least analytical reagent grade).

Double distilled or highly purified deionized water is used in this purpose.

The order of adding the chemicals should be maintained (as is described in

related literature) and each component is dissolved completely before adding
the next.

Appropriately labeled and stored at 40C!

 Preparation of Stock Solutions
Macronutrient Stock Solution
Usually prepared as 10X concentration.

All the macronutrients are added one after another except CaCl2.

The stock solution of CaCl2 should be prepared separately to avoid

precipitation.

Micronutrient Stock Solution

Generally prepared as 100X concentration.

Stock solution of copper and cobalt is prepared separately and then an

appropriate volume is added to the final solution.
 Preparation of Stock Solutions
Fe-EDTA Stock solution
Should be added in a fresh state.

Might be prepared as 100X solution.

Should be stored in an amber color bottle or a bottle covered with an

aluminium foil.

Stock Solutions for Vitamins and Growth Regulators

Growth regulators should be dissolved completely in an appropriate amount of
solvent (mentioned previously), and afterwards, water is added to match the
concentration (e.g. 10X).
Composition of the Stock Solutions of MS Medium
Preparation of
MS Medium
 Problem Solving!
Now prepare a 500 ml media having ¼ strength MS with 5mM BAP, 5.5 mg/L Kn
and coconut water 10%.

Stocks are MS Macro 10X, micro 20X, FeEDTA 10X, Organic 100X, BAP and Kn
5gm/100ml. If you need anything else then you add accordingly. BAP has a MW
of 225.3.

Sucrose and Myo-inositol is not mentioned in the question. These you need to
add. Consistency of the medium is also mentioned.
 Steps for Plant Tissue Culture
1. Preparation/Sterilization of instruments

2. Preparation and Sterilization of culture medium

3. Preparation of explant

4. Inoculation of Explant

5. Incubation for growth

• Callus formation
• Shoot induction
• Root induction

6. Acclimatization of plantlets (TCPs)

7. Transfer to pots
 Preparation of Explant
Different kinds of explants can be used for plant tissue culture, such as-

Petals

Leaves

Buds

Ovaries

Seeds

Anthers

Nodal segments
 Preparation of Explant
Explants are carefully taken from a disease free, healthy, and actively growing
plants, preferably having meristematic areas of high cellular activity.

Immediately after collection, the explants are placed in clean water to avoid
the entrance of microbes and other contaminants through the cut parts,

And also to avoid browning due to phenolic oxidation.

After that, the explants are taken for surface sterilization.

 Preparation of Explant: Surface Sterilization
The explant is cut into small pieces.

Placed in a petri plate w/ clean water.

Surface is brush-cleaned w/ a mild detergent (e.g. Tween 20)

Explants are then placed into a vessel containing a mild fungicide and/or an
antibiotic.

After a few minute, the explants are washed w/ clean and sterile DI H2O.

Explants are immersed in DI H2O and taken into the inoculation chamber.
 Inoculation of Explant
Inoculation means transferring plant specimen into the media under aseptic
conditions.

Laminar hood is prepared.

Explants are washed w/ 70% EtOH for a few seconds.

Treated w/ a mild solution of HgCl2 solution for a few minutes.

[***Use a mild solution for a longer duration rather than a strong solution for a
shorter duration***]

Explants are then washed several times with DI H2O to remove all traces of
HgCl2.
 Inoculation of Explant
All the apparatus are dipped in 70% EtOH, flamed using the lighted spirit lamp
and allowed to cool down.

The explants are then carefully picked w/ forcep and placed in the petri plate
(containing filter paper) to dry.

Surface of the explant exposed to HgCl2 are removed w/ the help of surgical
blade.

Each nodal segment is carefully inserted into the medium and care is taken to
avoid the explants from touching the rim of the flask.

The test tubes are labeled properly.

Then they are incubated.

 Incubation for Growth
The inoculated explant is incubated in the culture lab at optimum conditions of
16 hours light alternating w/ 8 hours of darkness.

Temperature: 25-270C

Relative Humidity: 40%

[***The conditions might differ with the plant species and the type of
explant***]

The cultures are monitored timely.

After a few days (5-15), all cultures with either bacterial or fungal contaminants
are removed.
 Incubation for Growth

After a period of time, you may get a large number of callus or shoots in a
single flask depending on the composition and ration of the growth regulators
in the culture medium.
 Shooting vs Rooting vs Callus Forming Media
Shoots are produced at high concentrations of cytokinin relative to auxin.

Roots were formed when the ratios were reversed.

Undifferentiated tissue or callus formed at hormone concentrations that are

optimal for callus growth, but not for shoot or root formation.

The values are different and should be optimized for individual plant tissue
culture technique.
Shooting vs Rooting vs Callus Forming Media

The values are different and should be

optimized for individual plant tissue culture
technique.
 Incubation for Growth

If callus is produced at the first place, they are then transferred to the shooting
media.

After a certain period of time, one ends up with a large number of shoots in a
culture flask.

Once the shoots have grown to a certain appreciable height of about 3-4cm,
they are transferred to rooting media and are rooted.

The rooted shoots have to hardened for acclimatization in the open.

 Acclimatization of TCPs
The Tissue Culture-raised Plantlets (TCPs) are transferred to greenhouse or
outdoor conditions where they are subjected to different types of shocks, like-

Temperature

Humidity

Nutrition

Carbon-di-oxide and

Air-flow shock
 Transferring to Pots

After rooting and growth of plantlets up to 3-4 inches, these are shifted to
other potting mixture containing garden soil, sand and decomposed farmyard
manure in ratio of 1:1:1.
 Transferring to the Field

After 45-60 days, these acclimatized plantlets can be shifted to the outdoor
conditions.

These plantlets are now ready for the harsh conditions effectively with very
low mortality.