1.

Macro elements
Macro elements are essential for satisfactory growth and morphogenesis of the plant.
Nitrogen (N): Component of nucleic acids, proteins, hormones, and coenzymes
In form of: NH4NO3 - KNO3
Phosphorus (P): Component of nucleic acids, proteins, hormones, and coenzymes, various
intermediates in respiration, photosynthesis and energy transfer
In form of: NaH3PO4.7H2O - KH2PO4 – NaH2PO4 – NH4H2PO4
Potassium (K): Cofactor that functions in protein synthesis; major solute functioning in water
balance; operation of stomata
In form of: KCl2 - KH2PO4 – KNO3
Calcium (Ca): Important in formation and stability of cell walls and in maintenance of
membrane structure and permeability; activates some enzymes; regulates many responses of
cells to stimuli
In form of: CaCl2.6H2O - Ca(NO3)2.4H2O
Magnesium (Mg): Component of chlorophyll; cofactor of many enzymes
In form of: MgSO4.7H2O

2. Micro elements
Manganese (Mn): Active in formation of amino acids; activates some enzymes; required for
water-splitting step of photosynthesis
Form: MnSO4.4H2O, concentration: 15-100M
Boron (B): Cofactor in chlorophyll synthesis; may be involved in carbohydrate transport and
nucleic acid synthesis
Form: H3BO4, concentration: 6-100M
Zinc (Zn): Active in formation of chlorophyll; activates some enzymes
Form: ZN(SO4).7H2O, concentration: 15-30M
Copper (Cu): Component of many redox and lignin-biosynthetic enzymes
Form: CuSO4.5H2O, concentration: 0.04-0.08M
Cobalt (Co): Component of vitamin B12
Form: CoCl2.6H2O, concentration: 0.1-0.4M

Iodine (I): KI, concentration: 2.5-20M

Molybdenum (Mo): Essential for nitrogen fixation; cofactor that functions in nitrate reduction
Form: (NH4)6Mo7O24.4H2O, concentration: 0.007-1M
NaMoO4.2H2O, concentration: 0.007-1M
Iron (Fe): Component of cytochromes; activates some enzymes, used in media by chelating with
EDTA (Fe_EDTA)

3. Organic elements
Media cultures are supplemented with vitamins because many plants cannot synthesize
adequate quantity of vitamins. They are required for the growth and differentiation of the
plants. It includes:
Concentration (mg/l)
Myo-inositol 100
Acid nicotinic (niacin) 0.5-1
Pyridoxin HCl (B6) 0.05-1
Thiamin HCl (B1) 1-5
Riboflavin (B2) 1-10
Panthothenate calci (B5) 0.5-2.5
Biotin (H) 0.01-1
Acid folic (M) 0.1-0.5
Tocopherol (E) 1-50
Myo-inositol is added in small quantities to stimulate cell growth of most plant species

Some media were supplemented with natural substances or extracts such as

Concentration
Coconut water 10-15%
Yeast extract 1-2 g/l
Casein hydrolysate 1-2 g/l
Bee milk 1-5 g/l
Malt 1-10 g/l
Potato extract 3-5%
Corn extract 3-5%
Banana 3-5%

They play a role in the growth enhancement of the plant. The drawback of these organic
extracts is, they vary in the quantity and quality of the growth-promoting factors.

4. Auxins:
There are wo types: natural which includes IAA and synthetic which includes IBA, 2,4-D and
NAA.
Functions:

 Stimulates the production of callus (at high concentration) and roots (at low
concentration)
 Causes extension of stem
 Stimulates of cell elongation, cell division in cambium tissue
  Auxin + cytokinin stimulate differentiation of phloem and xylem
 High concentration of auxin can induce somatic embryogenesis
The essential function of auxins and cytokinin is to reprogram somatic cells in a determine state
of differentiation. Reprogramming causes dedifferentiation and then redifferentiation into a
new pathway. Thus, a cell that had been destined to develop into part of a leaf might become
embryogenic, producing somatic embryo. The mechanism that auxin causes dedifferentiation is
not understood
High concentration of exogenous auxin can be toxic because they stimulate production of
concentrations of ethylene, which can cause growth inhibition

5. Cytokinins:
The various cytokinins used in culture media are BAP (6-benzyloaminopurine), 2iP, Kinetin,
Zeatin, and TDZ.
Cytokinin cause cell division, which can lead to shoot regeneration in vitro by stimulating the
formation of shoot apical meristems and shoot buds.
The cell division caused by cytokinins can produce undifferentiated callus
A high concentration of cytokinin will block root development
Cytokinins can cause release of shoot apical dominance, thereby stimulating growth of lateral
buds and resulting in multiple shoot formation

6. Gibberellins:
It is a member of tetracyclic diterpenoid carboxylic acids. Currently, about 20 GAs have been
identified as plant growth regulators, out of which, gibberellin A3 is commonly used in culture
media.
GA3 stimulates the shoot elongation or the conversion of buds into shoots
GA3 interferes with bud initiation at a very early stages of meristem formation and thereby may
reduce shoot production in vitro if given to plant tissue cultures at the shoot bud initiation
stage
It is necessary to optimize the stage-specific effects of GA3

7. Abscisic acid
ABA facilitates somatic embryo maturation. In certain species, embryos may not be mature
adequately for lack of ABA
Endogenous ABA functions similarly during seed development in monocotyledonous and
dicotyledonous plants and conifers
ABA induces the formation of LEA proteins found at late stage of embryogenesis in both
somatic and sexual embryos
ABA might stimulate some regeneration processes and, rarely, may also reduce the production
of somatic embryos
It can either stimulate or inhibit the callus growth depending on the species of the cultured
plant. It also stimulates shoot proliferation and inhibits the later stage of embryogenesis.

8. Polyamines
Promotes adventitious root and shoot formation, somatic embryogenesis.
Implicated in growth regulation, cell division and differentiation, and also in the plant response
to various sources of stresses
Some polyamines used in tissue culture media: putrescine, spermidine

9. Meristem culture techniques

Meristem and meristem tip culture are used to generate pathogen-eradiated shoots that
subsequently serve as propagules for in vitro propagation
Meristem culture
Culture of the apical meristematic dome alone from either terminal or lateral buds, for the
purpose of pathogen elimination, is termed meristem culture
In reality, true meristem culture is rarely used because isolated meristems of many species
exhibit low survival rates and increased chance of genetic variability following callus formation
and indirect shoot organogenesis
Pathogen elimination can often be accomplished by culture of relatively larger (0.2-0.5mm)
meristem tip explants excised from plants that have undergo thermo- or chemotherapy
Meristem tip culture
The meristem tip is comprised of the meristem + 1/2 subtending leaf primodia (fig6.2). This
procedure termed meristem tip culture.

10. Shoot-tip and node culture techniques

Although not the most sufficient procedure, propagation from axillary shoots has proven to be
a reliable method for the micropropagation of a large number of species
Depending on the species, 2 methods, shoot and node culture, are used. Both methods rely on
the stimulation of axillary shoot growth from lateral buds following disruption of apical
dominance of the shoot apex
Shoot culture
Shoot culture (shoot tip culture) refers to the in vitro propagation by repeated enhanced
formation of axillary shoots from shoot tip or lateral buds cultured on medium supplemented
with PGRs, usually a cytokinin
The axillary shoots produced are either subdivided into shoot tip and nodal segments that serve
as secondary explants for further proliferation or are treated as microcuttings for rooting
In some species, modified storage organs such as miniaturized tubers or corms (fig6.3) develop
under inductive culture conditions from axillary shoots or rhizomes and may serve as the
propagule for either direct planting or long-term storage
When either verified pathogen-free stock plants are used or when pathogen elimination is not a
concern, relatively large (1-20mm) shoot tip or lateral bud primary explants (fig6.2) can be used
for cultures
Adventages of using larger shoot tips include greater survival, more rapid growth response, and
the presence of more axillary buds
Node culture
Node culture, a simplified form of shoot culture, is another method for production from pre-
existing meristems
Numerous plants such as potato do not respond well to cytokinin stimulate of axillary shoot
proliferation observed in the micropropagation of many crops
Axillary shoot growth is promoted by the culture of either intact shoots (from meristem tip
cultures) positioned horizontally on the medium (in vitro layering) or single or multiple node
segments
Typically, single elongated unbranched shoots, comprised of multiple nodes, are rapidly
produced
These shoots (microcuttings) are either rooted or acclimatized to ex vitro conditions or
repeatedly subdivided into nodal cuttings to initiate additional cultures
Although node culture is the simplest method, it is associated with the least genetic variation
Stage 0: Donor plant selection and preparation
Stage I: Establishment of aseptic cultures
Stage II: Proliferation of axillary shoots
Stage III: Pre-transplant (rooting)
Stage IV: Transfer to natural environment