THE PRINCIPLES OF ASEPTIC CULTURE

IN PLANTS

SBT 2131

ASSOC. PROF. DR. AROKIARAJ

 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Plants growing in natural habitat (requirements)

In vitro plant cultures (requirements)

Nutrient Media:

Essential elements

Organic supplements

Source of carbon
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components:

Macronutrients, inorganic nutrients e.g. Nitrates,

Phosphorus, Potassium, Magnesium, Sulphate,
Calcium – present as salts in the various media (g/l;
or mM)

The source of nitrogen in the media: nitrates (NO3 in

the form of Ammonium Nitrate or Potassium Nitrate)

The source of potassium ions (K+) from potassium

nitrate or potassium dihdrogen phosphate
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components:

Micronutrients, inorganic nutrients e.g. EDTA,

Ferrous, Zinc, Boron, Iodide, Manganese, Molybdenum,
Cobalt, Copper (µg/l; mg/l; or µM).

Iron seems more critical; chelated forms of iron or zinc

are commonly used in culture media rather than iron
tartrate/citrate as they are difficult to dissolve and ppt in
medium.

EDTA-Fe chelate: as EDTA is not stable in medium

(EDTA complexes with Fe)
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Carbon or Energy source

Sugars, e.g. Sucrose, Glucose, Raffinose, Celibiose,

Mannitol, Fructose, Myo-inositol, Arabinose, Rhamnose,
Trehalose, Xylose, Xylitol, Sorbitol (g/l; %)

Plant Cells / tissues lack autotrophic ability

Sucrose is the most preferred carbon source

Glucose supports equally good growth (but fructose is

less efficient)

Sucrose on autoclaving is converted into glucose and

 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components:

Amino acids, e.g. Glycine, Casein hydrolysate (CH),

Argnine, Asparagine, Leucine, Serine, Proline, Glutamine
(mg/l; or mM)

Taken up rapidly by plant cells for stimulating cell

growth
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Organic supplements

Vitamins, Pyridoxine.HCl (Vitamin B6), Nicotinic acid,

Thiamine.HCl (Vitamin B1), Biotin, Folic Acid, Calcium
Pantothenate (Vitamin B5), Riboflavin, Ascorbic Acid
(mg/l; µM)

Generally the vitamins are added in the range of 1-

10mg/l
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Other Organic supplements

A variety of organic extracts are used: coconut water,

yeast, malt, banana extract and tomato extract

Significant effects rendered by coconut water (5-20%)

and casein (0.05-1.0%) for plant cell growth

Generally the use of natural extracts is avoided:

quality and quantity of growth-promoting constituents vary
(age of tissue from which the extract is derived, thereby
affecting reproducibility of results).

L-asparagine could effectively replace yeast extract or

 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Activated Charcoal (AC)

AC is generally acid-washed and neutralized before

addition to culture medium (0.5-3%)

AC also helps to reduce toxicity by removing toxic

compounds (e.g. phenolics) produced during culture and
permits unhindered cell growth

Sometimes the darkening of tissues occur during

culture, AC helps to reduce this effect couple with the
adsorption of inhibitory compounds.
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Growth Regulators

Four broad classes of growth regulators are used:

auxins, cytokinins, gibberrellins and abscisic acid

The growth and organogenesis of tissues become

feasible only on the addition of one or more of these classes
of hormones to a medium

The ratio of hormones required for root or shoot

induction varies considerably with the tissue, which seems
directly correlated to the amount of hormones synthesized at
endogenous levels within the cells of the explant
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Auxins

Media are supplemented with various auxins:

Indole 3-cetic acid (IAA),

1-napthaleneacetic acid (NAA),
Indole-3-butyric acid (IBA),
2,4-Dichlorophenoxyacetic acid (2,4-D) and
1-napthoxyacetic acid (NOA).

They are generally dissolved in ethanol or NAOH.

 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Auxins
IAA occurs naturally in plant tissues

Other auxins which have proved effective in plant cell

culture:

chlorophenoxyacetic acid (4-CPA),

ρ-chlorophenoxyacetic acid (PCPA),
4-amino-3,5,6- trichloropicolinic acid (picloram),
3,6-dichloro-2-methoxybenzoic acid (dicamba)

Common feature of auxin: includes cell division, and in

nature, this group of hormones are involved with such
activities as elongation of stem, internodes, apical
dominance, abscission and rooting.
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Cytokinins

These are adenine derivatives, mainly concerned with

cell division, modification of apical dominance and
shoot differentiation in tissue culture.

Most frequently used cytokinins:

6-benzylaminopurine (BAP),
6-gama-gama-dimethylaminopurine (2-ip),
Kinetin and
Zeatin.
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Cytokinins

Zeatin and 2-ip are naturally occurring cytokinins while

BA and kinetin are synthetically derived cytokinins. They
are generally dissolved in dilute HCl or NAOH.

The ratio of auxins and cytokinins is important with

respect to morphogenesis on the cell culture medium.

For callus initiation, embryogenesis and root initiation

the requisite ratio of auxins to cytokinin is high, while the
reverse leads to axillary and shoot proliferation.
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Gibberellins and Abscisic acid

GA3 is the most common gibberellin used of more than 20

known gibberellins.

It promotes callus growth and induces embryogenesis.

It also induces stunted plants to elongate.

Abscisic acid (ABA) stimulates or inhibits callus growth

depending on the species, also promotes distinct
developmental pathways (somatic embryogenesis).
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Ethylene

Ethylene is a gaseous, naturally occurring, plant growth

regulator.

Associated with controlling fruit ripening.

Some plant cell cultures produce ethylene, which can inhibit

the growth and development of the culture.
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Other substances (as hormones)

Jasmonates (jasmonic acid and its methyl ester), play a

role in embryogenesis, differentiation, root formation and
breaking of seed dormancy.

Salicyclic acid promotes flowering and inhibits ethylene

biosynthesis.

Brassinosteroids play a role in shoot elongation, inhibition

of root growth.
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Solidyfying Agents

Commonly used for preparing semi-solid or solid tissue

culture media: Agar, a polysaccharide from seaweeds.

Normally 0.5 to 1.0 % agar is used in the medium to firm

gel at required pH.

Others used are: phytagel and gel-rite (clear gelling

agents are valuable aids for detecting contamination)
 BASIC MEDIA COMPONENTS
FOR PLANT CELL CULTURE
Media components: Solidyfying Agents

Plant Cells and tissues require optimum pH for growth

and development.

The pH of a medium can be adjusted to the requirement

of the experiment.

The pH affects uptake of the ions and for most of the

culture media, pH 5.0 to 6.0 before sterilization is
considered optimal.
 Reference Book
(Available in Kuantan Campus Library)

Plant Cell Culture, D.E. Evans, J.O.D. Coleman and A. Kearns

BIOS Scientific Publishers, London and New York

ISBN 1 85996 320 x