Plant Tissue Culture
Paper IV Unit III
T.Y.B.Sc. Biotechnology
By
Mr. Ketan Thatte
Introduction
What is Plant Tissue Culture?
• Plant tissue culture is the aseptic (free from
microorganism) culture of any plant part in vitro
on an artificially prepared nutrient media and
under controlled conditions.
• Plant tissue culture relies on the fact that any
plant cells have the ability to regenerate into a
whole plant i.e. Totipotency.
• Single cells, Protoplasts (plant cells without
cell walls), Leaf material, nodal /internodal
segments of stem or roots can often be used to
generate a new plant on culture media.
• It has advanced the knowledge of fundamental
botany, especially in the field of agriculture,
horticulture, plant breeding, forestry, somatic
cell hybridization, phytopathology and
industrial production of plant metabolites.
Terminology in PTC
•
Auxin---A group of plant growth regulators that
promotes callus growth, cell division, cell
enlargement, adventitious buds, and lateral rooting.
E.g. 2,4-Dichlorophenoxy acetic acid (2,4-D), Indole3-butyric acid (IBA), α-Naphthalene acetic acid
(NAA)
•
Cytokinin---A group of plant growth regulators that
regulate growth and morphogenesis stimulate cell
division. Zeatin, 6-furfurylaminopurine (kinetin) and
6-benzylaminopurine (BA or BAP).
•
Gibberellins---A plant growth regulator that
influences cell enlargement. Gibberellic Acid (GA3).
•
•
•
•
•
Explant---Tissue taken from its original site and
transferred to an artificial medium for growth
maintenance.
Callus---An unorganized, proliferate mass of
differentiated plant cells, a wound response.
Totipotency---A cell characteristic in which the
potential for forming all the cell types in the
organism are retained.
Micropropagation---In vitro clonal propagation of
plants from shoot tips or nodal explants, usually
with an accelerated proliferation of shoots during
subcultures.
Clonal Propagation---Asexual reproduction of
plants that are considered to be genetically uniform
and originated from a single individual or explant.
•
•
•
•
Shoot Apical Meristem---Undifferentiated tissue,
located within the shoot tip, generally appearing as a
shiny dome-like structure, distal to the youngest leaf
primordium and measuring less that 0.1 mm in length
when excised.
Somaclonal Variation---Phenotypic variation, either
genetic or epigenetic in origin, displayed among
somaclones.
Somaclones---Plants derived from any form of cell
culture involving the use of somatic plant cells. Or
plants produced asexually from a single source plant.
Subculture---This is the process by which the tissue
or explant is first subdivide, then transferred into
fresh culture medium.
History
• 1902: Haberlandt demonstrated the ‘Totipotency’.
• 1902: Haberlandt reported single palisade cell culture
from leaves in knop’s salt solution
• 1904: Hanning practiced ‘Embryo culture’ of
Crucifers.
• 1925: Laibach recovered ‘Hybrid progeny from
interspecific cross in Linum.
• 1926-34: Discovery of IAA and role of Vit. B in plant
growth and root development.
• 1939: Gautheret, white and Nobecourt established
first callus culture from cambium tissue.
• 1944: Skoog reported ‘shoot bud differentiation’ in
tobacco pith tissues.
• 1955: Miller et al. discovered ‘kinetin’ enabled callus
culture from differentiated tissues.
• 1957: Skoog and Miller proposed ‘root-shoot
differentiation’ in vitro by auxin-cytokinin ratio.
• 1959: Braun regenerated first plant from mature plant
cell.
• 1958-59: Reinert and Stewart developed ‘somatic
embryos’ from carrot.
• 1960: Cocking isolated ‘Plant protoplast’ using cell wall
digesting enzymes. Followed by Bergmann for
‘Suspension culture’.
• 1962: Murashige and Skoog developed first
complete nutrient medium.
• 1964: Maheshwari and Guha produced ‘Haploid
plants from pollen grains of Datura.
• 1966: Nitch and Nitch isolated microspores of Tobacco
to produce haploid plants via ‘Anther culture’.
• 1972: Carlson et al. produced first somatic hybrid by
fusing protoplasts of Nicotiana glauca and N.
langsdorfii.
• 1988: Levin et al. Developed bioreactor system for
embryogenic and organogenic cultures of several
plants.
• 1990: Application of PTC techniques for the production
of secondary metabolites from Medicinal and
Aromatic plants.
Culture Medium for Plant Tissue Culture
• An artificially prepared liquid or gelatinous substance
containing nutrients in which excised plant tissues or
organs are cultivated is called as Culture Medium.
• Chemically Defined: Composition and concentration
of each nutrient is exactly known.
• Chemically Undefined: Composition and
concentration of nutrient is not exactly known due to
addition of organic supplements.
• Macro Inorganic Salts: It contains the salts that are
needed in higher amounts. These include Nitrogen,
Phosphorus, Sulphur, Magnesium, Calcium and
Potassium.
• Micro Inorganic Salts: It contains the salts that are
needed in trace amounts. These include Boron,
Manganese, Zinc, Iodine, Molybdenum, Copper and
Cobalt.
• Iron Source: It is supplied as a combination of Ferric
salt and Disodium salt of Ethylene diamine tetra acetic
acid (Fe-EDTA).
• Amino Acids: Most commonly used amino acid is
Glycine.
• Vitamins: This includes Meso inositol, Nicotinic acid,
Pyridoxine HCL, Thiamine HCL.
• Carbon Source: Carbohydrates is supplied in the form
of Sucrose.
• Phytohormones: Generally Auxins and Cytokinins are
required singly or in combination to initiate and
maintain cells.
• Auxins: IAA, IBA, NAA, 2,4-D.
• Cytokinins: BAP, Kinetin, Zeatin, 2iPA, TDZ.
• Agar: The culture medium is solidified with 0.8% agar.
A semisolid medium is the one in which agar
concentration is less than 0.8%. A medium devoid of
agar is called liquid medium.
Preparation of Stock Solution:
• Macro salts: Stock is prepared as 20X.
• Micro salts: Stock is prepared as 100X.
• Iron EDTA: Stock is prepared as 50X
• Vitamins & Growth Hormones: 1mg/ml. concentration.
• Amino acids: 1mg/ml.
• Sucrose and Agar agar is added as per the requirement.
• Preparation of 1 Liter of MS Medium:
Addition of stock solutions is done as follows:
R x T/G
where R is required concentration,
G is given concentration, T is total volume
Plant Nutrition
VITAMINES
• They function as catalyst in enzyme reaction
• Thiamine required for carbohydrate metabolism and
synthesis of amino acid
• Other examples: Nicotinic acid, Pyridoxine HCl,
Biotin, Folic acid, Riboflavin, pantothenic acid, Paraamino benzoic acid, Ascorbic acid.
MYO-INOSITOL
• It is a sugar alcohol, improves in-vitro responses
AMINO ACIDS
• Functions in purine synthesis and is a part of porphyrin
ring of chlorophyll .
CARBOHYDRATES
• Sugars added in the medium act as an energy and carbon
source
• Act as osmoticum. Osmotic potential have an important
effect on in-vitro response
PLANT GROWTH REGULATORS
• Auxins: It induces cell elongation, cell division and
somatic embryogenesis. At higher concentration, auxin
promotes root formation.
• Cytokinins: Essential to promote cell division, lateral
bud development and cytokinesis. At higher
Concentration, they promote shoot formation.
• Gibberellins: Influences stem elongation and
flowering. Helps in seed germination by breaking the
dormancy
Element
Role
Nitrogen (N)
Helps plant to synthesize complex
organic molecules
Important role in osmoregulation and
anion-cation balance of plant.
Phosphorous (P)
Essential element in photosynthesis and
respiration
Affects plant maturation and root growth
Potassium (K)
Essential for normal cell division and
promotes meristematic growth
Involved in maintaining the turgor
pressure of its cells
In opening and closing of stomata
Accumulation and translocation of
carbohydrates
Sulphur (S)
Present in Cysteine and Methionine
which are precursor for vitamins and
coenzymes
Calcium (Ca)
Ca- pectate- an integral component of
cell wall
Element
Roles
Manganese (Mn)
In Nitrate assimilation
Involved in oxidation-reduction process
in photosynthetic electron transport
Boron (B)
Helps in absorption of calcium and
assists in pectin formation of cell wall
Involved in cell division, differentiation,
maturation, respiration
Associated in pollen germination
Molybdenum (Mo)
Its deficiency results in deformation of
flowers and leaves dieback
Zinc (Zn)
Activates auxin production
Helps in chlorophyll formation and
prevents chlorophyll destruction
Copper (Cu)
Involved in carbohydrate metabolism,
nitrogen fixation and in oxygen
reduction
Cobalt (Co)
Essential for Nitrogen fixing bacteria
Component of Vit. B12 and enzymes,
needed to form aromatic compounds
Iron (Fe)
Essential for chlorophyll formation