Laboratory organization

and requirements of PTC

 (A) Plant Material (B) Tissue culture medium

(1a) Sterilization (2) Sterilization

Transfer

Explants

(1b) Sterilization

Sterilization, either at
stage “1a” or “1b”
 Instruments
Hands

(2 or 4) Sterilization
(3) Sterilization
Laminar Air flow / Clean Bench
 Plant Growth Room / Incubator

Control system for: Light, Temp, and humidity

 Direct use through tissue culture

Transfer to field

Transfer to field

Acclimatization Green house

Why acclimatization is required??
(Photosynthesis and root absorption)
 Plant Tissue culture Media (PTM)

PTM is an replica of soil where plant can grow

Major Constituents of Plant Growth Medium

Composition of Media:

The composition of the culture media is primarily dependent on three parameters:

1. The particular species of the plant.

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

3. What tissue culture response you are looking for?

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.
The culture media usually contain the following constituents

1. Inorganic nutrients

2. Carbon and energy sources

3. Organic supplements

4. Solidifying agents

5. Growth regulators

6. pH of medium
A. Inorganic Nutrients:

The inorganic nutrients consist of

Macronutrients (concentration >0.5 mmol /L)

Micronutrients (concentration <0.5 mmol /L).

A wide range of mineral salts (elements) supply the macro- and micronutrients. The inorganic

salts in water undergo dissociation and ionization. Consequently, one type of ion may be

contributed by more than one salt.

For instance, in MS medium, K+ ions are contributed by KNO3 and KH2PO4 while NO3– ions
come from KNO3 and NH4NO3.
Macronutrients:

The essential elements in plant cell or tissue culture media include, besides C, H and
O,

Macroelements: nitrogen (N), phosphorus (P), potassium (K), calcium (Ca),

magnesium (Mg) and sulphur (S) for satisfactory growth and morphogenesis.

Concentration:

Inorganic Nitrogen: at least 25-60 mM of inorganic nitrogen for satisfactory plant cell
growth.

Potassium (20 – 30 mM) is required for cell growth of most plant species. Potasium in
used in the form of nitrate and / or chloride salts

The optimum concentrations of P, Mg, S and Ca range from 1-3 mM if other

requirements for cell growth are provided
 Nitrogen

Nitrogen = 25-60 mM

Nitrogen: Gives better results when both Nitrate and ammonium together as nitrogen
source.
Nitrate: 20-25 mM,
Ammonium = 2-20 mM; optimum for most plants = 8 mM

Cells can grow on a culture medium containing ammonium as the sole nitrogen
source if one or more of the TCA cycle acids (e.g., citrate, succinate, or malate)
are also included in the culture medium at concentrations of approximately 10
mM
Micronutrients:

Iron (Fe), 1 µM
manganese (Mn), 20 – 90 µM
zinc (Zn), 30 µM
boron (B), 25 – 100 µM
copper (Cu) 0.1 µM
molybdenum (Mo). 1 µM

Iron is usually the most critical of all the micronutrients. The element is used as either
citrate or tartarate salts in culture media, however, there exist some problems with
these compounds for their difficulty to dissolve and precipitate after media
preparation. There has been trials to solve this problem by using ethylene
diaminetetraacetic acid (EDTA)-iron chelate (FeEDTA) to enhance solubility,.

Cobalt (Co) and iodine (I) may be added to certain media, but their requirements for
cell growth has not been precisely established.
Function of Inorganic nutrients:
1. Concentration of micronutrient “A” in the medium is 0.488 mmol/L and
Concentration of “B” is 0.325 mmol/L. Answer which one is Macronutrients?

2. Which micronutrient is most critical for plant growth?

3. Fe-EDTA is preferred over FeCl3 in preparing plant medium, Why?

B. Carbon / Energy Source:

Sucrose is the most frequently used carbon source in plant medium. (Concentration range
(2-5 %; w/v)

Other carbon source: Glucose, lactose, galactose, maltose and starch

Glucose is more effective than fructose

considering that glucose is utilized by
the cells in the beginning, followed by
fructose.

It was frequently demonstrated that

Glucose Fructose
autoclaved sucrose was better for growth
than filter sterilized sucrose.

WHY????
It was frequently demonstrated that autoclaved sucrose was better for growth than
filter sterilized sucrose.

ANSWER: during autoclave sucrose is broken down into glucose and fructose

Sucrose was reported to act as morphogenetic trigger in the formation of auxiliary

buds and branching of adventitious roots

Glucose: NOT Autoclavalble; Filter Sterilize

Why sucrose is preferred over glucose as carbon source in most of the plant

medium ?
C. Organic Supplements:

1. Vitamins and MYO-INOSITOL

2. Amino Acids
3. Undefined organic supplements

Vitamins

Required by plants as catalysts in various metabolic processes. They may act as

limiting factors for cell growth and differentiation when plant cells and tissues are
grown In vitro (Tissue culture)

Essential Vitamin Use Concentration (mg/L)

Thiamin (B1) For growth 0.1 - 10
Nicotinic acid Growth (not essential for all) 0.1- 5
Pyridoxine (B6) Growth (not essential for all) 0.1- 10
Other vitamins such as biotin, folic acid, ascorbic acid, pantothenic acid, tocopherol

(vitamin E), riboflavin, p-amino-benzoic acid are used in some cell culture media

however, they are not growth limiting factors.

MYO-INOSITOL

NOT a vitamin but a carbohydrate, myo-inositol is

added in small quantities to stimulate cell growth of
most plant species. Myo-inositol is believed to play a
role in cell division because of its breakdown to
ascorbic acid and pectin and incorporation into
phosphoinositides and phosphatidyl-inositol. It is
Myo-Inositol
generally used in plant cell and tissue culture media
at concentrations of 50-5000 mg/L.
Amino acids

Amino Acid: Mostly synthesized by plants.

Callus / protoplast culture: Requires additional amino acid supplement for proper growth.
Easy source of nitrogen: Amino acid provides easily metabolized source of nitrogen than
inorganic nitrogen.
Casein hydrolysate, L-glutamine, L-asparagine and adenine are frequently used as sources
of organic nitrogen in culture media
Casein hydrolysate = 0.25-1 g/L
glycine = 2 mg/L
glutamine = up to 8 mM
asparagine = 100mg/L
L-arginine and cysteine = 10 mg/L
L-tyrosine = 100mg/L

Tyrosine has been used to stimulate morphogenesis in cell cultures but should
only be used in an agar medium
Organic acids:

Addition of Krebs cycle intermediates such as citrate, malate, succinate or fumarate allow

the growth of plant cells. Pyruvate also enhances the growth of cultured cells.
UNDEFINED ORGANIC SUPPLEMENTS

Some media were supplemented with natural substances or extracts such as protein

hydrolysates, coconut milk, yeast extract, malt extract, ground banana, orange juice and

tomato juice, to test their effect on growth enhancement.

Explanation of the mode of action of activated charcoal was based on adsorption of

inhibitory compounds from the medium, adsorption of growth regulators from the

culture medium or darkening of the medium

The presence of 1% activated charcoal in the medium

was demonstrated to largely increase hydrolysis of
sucrose during autoclaving which cause acidification of
t h e c u l t u r e m e d i u m .
D. Solidifying agent:

Solidifying agent: Agar, a polysaccharide obtained from seaweeds

Advantages of Agar: it easily melts in a temperature range 60-100oC and solidifies at

approximately 45oC and it forms a gel stable at all feasible incubation temperatures. Agar
gels do not react with media constituents and are not digested by plant enzymes.

Concentration: 0.8 – 1% Agar is derived from the polysaccharide agarose, which

forms the supporting structure in the cell walls of certain
species of algae, obtained from Red algae:
Gelidium and Gracilaria

Gelidium
 Phytagel: An agar substitute produced from a bacterial fermentation composed of
glucuronic acid, rhamnose and glucose. It produces a clear, colorless, high strength gel
which aids in detection of microbial contamination as well as helps in proper
visualization of root associated growth.

Agar Phytagel

Gelrite at 0.2% can be used for solidification of media. Gelrite gels are remarkably
clear in comparison to those formed by agar. Gelrite requires both a heating cycle and
the presence of divalent cations (Mg++ or Ca++) for gelation to take place.
Plant Tissue
Culture medium
White’s medium:
This is one of the earliest plant tissue culture media developed for root culture

MS medium:
Murashige and Skoog (MS) originally formulated a medium to induce organogenesis,
and regeneration of plants in cultured tissues. These days, MS medium is widely used
for many types of culture systems.

B5 medium:
Developed by Gamborg, B5 medium was originally designed for cell suspension hairy root
and callus cultures. At present with certain modifications, this medium is used for
protoplast culture.

N6 medium:
Chu formulated this medium and it is used for cereal anther culture, besides other
tissue cultures.

Nitsch’s medium:
This medium was developed by Nitsch and Nitsch and frequently used for anther cultures
Synthetic and natural media:

When a medium is composed of chemically defined components, it is referred to as a

synthetic medium. On the other hand, if a medium contains chemically undefined
compounds (e.g., vegetable extract, fruit juice, plant extract), it is regarded as a natural
medium. Synthetic media have almost replaced the natural media for tissue culture.

Expression of concentrations in media:

The concentrations of inorganic and organic constituents in culture media are usually

expressed as mass values (mg/L or ppm). However, as per the recommendations of the

International Association of Plant Physiology, the concentrations of macronutrients

should be expressed as mmol/L and micronutrients as µmol/L

E. Growth Regulators:

Plant growth regulators are important in plant tissue culture since they play vital roles
in stem elongation, tropism, and apical dominance. They are generally classified into
the following groups;

Auxins,

cytokinins,

gibberellins

abscisic acid.

Moreover, proportion of auxins to cytokinins determines the type and extent of

organogenesis in plant cell cultures.
AUXINS

The common auxins used in plant tissue culture media include:

indole-3- acetic acid (IAA) (only natural auxin occurring in plant)

indole-3- butric acid (IBA),
2,4-dichlorophenoxy-acetic acid (2,4-D)
naphthalene- acetic acid (NAA).

Other synthetic auxins used in culture media such as

4-chlorophenoxy acetic acid or p-chlorophenoxy acetic acid (4-CPA, pCPA),
2,4,5-trichloro-phenoxy acetic acid (2,4,5 T),
3,6-dichloro-2-methoxy- benzoic acid (dicamba)
and 4- amino-3,5,6-trichloro-picolinic acid (picloram)
Chemical Structure of common auxins
Chemical Structure of common auxin-like-compound
How to prepare Auxin stock solution
FUNCTION of auxins

In tissue cultures, auxins are usually used to stimulate callus production and cell

growth, to initiate shoots and rooting, to induce somatic embryogenesis, to

stimulate growth from shoot apices and shoot

stem culture
CYTOKININS

The common CYTOKININS used in plant tissue culture media include:

BAP (6-benzyloaminopurine)
2iP (6-dimethylaminopurine)
kinetin (N-2-furanylmethyl-1H-purine-6-amine)
Zeatin (6-4-hydroxy-3-methyl-trans-2-butenylaminopurine)
TDZ (thiazuron-N-phenyl-N-1,2,3 thiadiazol-5ylurea).

Zeatin and 2iP are naturally occurring cytokinins and zeatin is more effective.

In culture media, cytokinins proved to stimulate cell division, induce shoot

formation and axillary shoot proliferation and to retard root formation

Stock solution: Prepare in DMSO, 1N HCL, stable, autoclavable.

Chemical Structure of common Cytokinins
FUNCTION of cytokinins

Cytokinins also have a clear effect on cell division. Often used to stimulate growth and

development, they usually promote cell division if added together with an auxin. Auxins

favour DNA duplication and cytokinins enable the separation of chromosomes.

Cytokinins have a clear role in organogenesis where they stimulate bud formation.

They are antagonistic to rhizogenesis.

Stock solutions of IAA and kinetin are stored in amber bottles or bottles covered with a
black paper and kept in the dark since they are unstable in light
Gibberellins

Gibberellins are normally used in plant regeneration. GA3 is essential for meristem

culture of some species. In general, gibberellins induce elongation of internodes and

the growth of meristems or buds in vitro. In its absence, the culture appears globular,

due to the accumulation of nodes. Gibberellins usually inhibit adventitious root as well

as shoot formation. During organogenesis, gibberellins are antagonistic. They seem to

oppose the phenomenon of dedifferentiation.

Abscisic acid

Abscisic acid is an important growth regulator for induction of

embryogenesis. Abscisic acid was identified in 1965 and since then, has been

found in all plants. This is a growth inhibitor, which seems to be synthesized

when a plant is under difficult conditions. It has a favourable effect on

abscission.
Anti-browning compounds in the media

Many plants are rich in polyphenolic compounds. After tissue injury during
dissection, such compounds will be oxidized by polyphenol oxidases and the tissue
will turn brown or black. The oxidation products are known to inhibit enzyme activity,
kill the explants, and darken the tissues and culture media, a process which severely
affects the establishment of explants.

Anti-browning agents:
1. Activated charcoal at concentrations of 0.2 to 3.0% (w/v)

2. It can adsorb toxic brown/black pigments and also stabilize pH. Besides activated
charcoal, polyvinylpyrolidone (250-1000 mg/l), citric acid and ascorbic acid (100 mg/l
each), are also used to prevent oxidation of phenols.
pH of the media

Nutrient medium pH ranges from 5.0 to 6.0 for suitable in vitro growth of explant. pH

higher than 7.0 and lower than 4.5 generally stops the growth and development. The

pH of the medium changes during autoclaving. It generally falls by 0.3 to 0.5 units after

autoclaving. If the pH falls appreciably during plant tissue culture (the medium becomes

liquid), then a fresh medium should be prepared. One should know that a starting pH of

6.0 could often fall to 5.5 or even lower during growth. pH higher than 6.0 gives a fairly

hard medium and a pH below 5.0 does not allow satisfactory gelling of the agar.

***If your growth regulator is dissolved in 1N NaOH of 1N HCL, then add them
prior to autoclave. If they are heat labile then adjust pH as per increase /
decrease in pH due o addition of growth regulator.
pH adjustment of media

1. To increase pH: drop by drop addition of 1N NaOH / 1N KOH

2. To decrease pH: Drop by drop addition of 1N HCL

3. Do not use H2SO4, HNO3 for pH adjustment

4. Always use long stirring for pH adjustment (See video)

5. Add growth regulator before pH adjustment

6. Add agar and melt at the end before autoclaving

1. Why one not use H2SO4, HNO3 for plant pH adjustment?
2. Why growth regulator should be added before pH adjustment?
3. Why continuous stirring is required for pH adjustment?
 General methodology for preparation of medium (MS Medium)

These concentrated solutions are called stock solutions.

Simple stock solutions comprise only one constituent at a time. Complex stock
solutions comprise several chemicals. Stock solutions of macro and
micronutrients, vitamins and growth regulators are prepared in distilled or high
purity demineralized water. Chemicals should be of the highest grade.

Macronutrient stock solution(s): Usually, the stock solution of macronutrients is

prepared as 10x. Dissolve all the macronutrients one by one except (CaCl2 for
macronutrient stock solution. The stock solution of CaCl2 should be prepared
separately. Another way is to dissolve the different macronutrients one after the other
and CaCl2 is dissolved separately and later added to the rest of the stock solution in
order to avoid precipitation.
Micronutrient stock solution: A stock solution of all the micronutrients with 100x is
generally prepared. Since copper and cobalt are required in very small quantities, it is
preferable to first make a separate stock solution of these two salts (100x) and then an
appropriate volume can be pipetted and put into the main micronutrient stock
solution. These nutrient solutions can be dispensed in plastic bags with zipper seals
and stored frozen.

Iron-EDTA: Iron EDTA should be added fresh. If stock solution (100x) is prepared,
then it should be stored after autoclaving in an amber bottle or a bottle covered
with an aluminium foil.
Vitamins and growth regulators stock solutions: These are simple stock
solutions. All the growth regulators are not soluble in water. The compound
should be dissolved in a few ml of solvent and then water is slowly added to
make the requisite volume. Concentrations of compounds can be taken as mg/l
or in molarity.
Sucrose = 3% w/v

Agar = 0.8% w/v

Add the component into a beaker according to the list : Macronutrients, micronutrients iron-
EDTA, vitamins, myo-inositol, growth regulators (if thermostable and autoclavable), organic
supplements, sucrose etc., by using the correctly sized graduated cylinders or pipettes or
balance.

2. Water is added to just below the final volume (e.g. 800 ml volume for one litre medium)

3. pH of the medium is adjusted to the required value (e.g. pH 5.8 for MS) by adding drop-wise
while stirring 1N KOH or 1N HCl.

4. Required quantity of agar or any other gelling agent is added while the medium is being
stirred.

5. The solution to brought to the final volume, i,e 1 Litre and heated with continuous stirring
until all the agar is dissolved and the solution becomes transparent.
6. The medium is dispensed in glass or polypropylene vessels and plugged with cotton plugs.

7. Culture medium is sterilized in an autoclave for 20 min at 121oC at 15 psi (105 kPa).
 If the medium contains heat-labile substances :F

a. steps 1- 5 are followed except for the addition of heat labile substances.
b. Culture medium is sterilized as such in a big Erienmeyes flask without
dispensing in vessels in an autoclave for 20-25 min at 121oC at 15 psi (105
kPa).

c. the thermolabile compound solutions are filter sterilized using millipore or

any other filter assembly using 0.22 μm filter.

Bacterial size = 1-10 uM

Virus = 100 nm
d. After autoclaving, the medium is kept in a laminar airflow hood and
allowed to cool to a temperature of around 50oC. The requisite quantity of
the compound is added to the medium with the help of micropipettes while
the mediuim is being stirred.

e. the medium is dispensed into sterile containers (generally sterile petri

dishes) under the hood of laminar airflow, provided the neck of the
Erlenmeyer flask is passed over a flame before the medium is poured from
it.
Basic calculations

Units in weight It is represented as milligram per litre (mg/l)

10-6 = 1.0 mg/l or 1 part per million (ppm)
10-7 = 0.1 mg/l.

Molar concentration A molar solution (M) contains the same number of grams
of substance as is given by molecular weight in total volume of one litre.

1 molar (M) = the molecular weight in g/l

1 mM = the molecular weight in mg/l or 10-3 M

1. 1mM NaCl = ? Mg/L

Conversion from milli molar (mM) to mg/l
For example,
molecular weight of auxin 2,4-D = 221.0
1M 2,4-D solution consists of 221.0 g per litre
1 mM 2,4-D solution consists of 0.221 g per litre = 221.0 mg per litre
1 μM 2,4-D solution consists of 0.000221 g/l = 0.221 mg/l
Sterilization Techniques
Chemical Sterilization

Agent Conc Treatment Remarks

time
Sodium 1 – 1.5% 4-30 min Very good
hypoclorite
Calsium 9 -10% 4-30 min Very Good
hypochlorite
Hydrogen 10 -12% 5 -15 min Good
peroxide
Mercuric 0.01 – 1% 2 – 10 min Good*
chloride * carcinogenic
Antibiotics 5-50 mg/L 30 to 60 min Good
Expensive
 Chemical Sterilization

Commercial solution 5-6%

Chemical Sterilization

Take plant tissue

Wash is tap water (3-4 times)

Wash in 70% (v/v) Ethanol for 30 sec to 2 min

Wash in 70% (v/v) Ethanol for 30 sec to 2 min

Or wash with few drops of detergent tween 20

Wash in water (2 times)

20% sodium hypochlorite (1% chlorine) for 5 -10 min *

Wash in sterile water (5-6 times)

* Need to optimize concentration and time depending on tissue

Chemical Sterilization

* How you can quickly optimize NaOCl treatment time?

Flame Sterilization

After sterilization for 2-3 min,

instruments are soaked into 70-80%
alcohol. This should be done repeatedly.
Stem Sterilization

Vol. Media Time Temp Pressure

20-50 ml 20 min 121 degree C 15 psi
50-500 ml 25 min 121 degree C 15 psi
500-1000 ml 35 121 degree C 15 psi

1. Autocleve should not be tightly packed

2. Bottle cap should be loose
3. Media volume in flask

1. For instrument use dry autoclave cycle

2. 3h heating at 160 - 180 degree C
3. Instrument should be packed with aluminum foil/
Alcohol Sterilization

Sterilization of hands. 70% Ethanol. Also clean laminar flow with 70%
ethanol before and after use.

70% Ethanol

* Why 70% ethanol Not 100%?

Ultraviolet Sterilization

Laminar Airflow
Dispensing of media under laminar flow

UV C light: 100 – 280 nm