Plant tissue culture

Plant tissue culture: Plant tissue culture has a great significance in plant biotechnology specially in the
crop improvement programmes.

Tissue culture: the process of in-vitro culture of explants (pieces of living differentiated tissues) in
nutrient medium under aseptic conditions.

Cell Culture: Denotes the in-vitro culture of single or a few cells.

Organ Culture: This term is used for in-vitro culturing of organs like embryo, root or shoot apices.
Suspension Culture: Defined as the culture of cell and cell aggregates suspended in a liquid medium.
Ex plant: The excised piece of differentiated tissue or the organ which is used for culture is called as
explant. e.g., embryos, young leaf, bud, etc.
Callus: The undifferentiated mass of cells is referred to as callus. The cells of callus are meristematic in
nature.
Explant: an excised part of differentiated tissue or organ or plant part. Eg: Leaf, Stem,Root,Seeds etc.
Totipotency: it is the inherent capacity of a plant cell to convert into callus or organ or plant part or
whole plant.
Totipotecy is the ability of change to meristematic stateand differentiate into whole plant.
Totipotecy= dedifferentiation+redifferentiation whole plant.
Dedifferentiation: the phenomenon of mature cells changing to meristematic state to produce callus.
Redifferentiation: the ability of callus cells to differentiate into plant organ or a whole plant.

History of Plant Tissue Culture:

The ability of the plant material to grow and divide invitro has been known for many years.
1. In 1860 SACH & KNOP tried to use inorganic salt for the growth of tissue artificially.
2. In 18078 VOCHTUNG observed that the cell along the stem length were capalble of generating
roots or shoots.
3. In 1892 KLERKER first attempt to isolate protoplast.
4. In 1902German botanist Gottleib Haberlandt was the first person to make attempts for plant
tissue culture, i.e., he developed the concept of in-vitro culture of plant cells and is aptly regarded
as the father of tissue culture. Thereafter, there happened some dramatic advances in tissue
culture techniques.
5. In 1904 HANNING established embryo culture of Cochleria using mineral salt and sugar
solution.
6. In 1922 Haberlandt students KOTTE & ROBBINS succeded in vitro cultivation of root tips. i.e.
Zea,Pea.
7. In 1934 WHITE first permanent root culture ( lycopersicum) and GAUTHERET first permanent
callus culture using Vitamin B, Auxins (Dacus) were demonstrated.
8. In 1942 GAUTHERET Studied thje secondary metabolite in cultured plants.

Some of the early classical contributions in the field of plant tissue culture are tabulated below:
S.No. YEAR Name of the Achievement On Species
Research Person
1 1892 KLERKER First attempt to
isolate protoplast
2 1902 HABERLANDT First attempt of Tradescantia
in-vitro culture of
plant cell

Kotte mahendar,
M.Pharmacy, (Ph.D), Dept. of Pharmacognosy & Phytochemistry.
Plant tissue culture
1904 HANNING Established Cochleria
Embryo culture
4 1922 KOTTE & Invitro culturing Zea,, Pisum.
ROBBINS of roots.
5 1925 LAIBACH Zigotic embyo Linium spp.
culture
6 1934 WHITE First permanent Lycopercium
root culture
7 1934 GAUTHERET First permanent Dacus
callus culture
using Vit.B,
Auxins
8 1939 GAUTHERET, Succesfull -
WHITE & establishment of
NOBECOURT indefinite callus
culture
9 1941 BRAUN Culture of Crown -
Gall Tissue
10 1942 GAUTHERET Observation of -
secondary
metabolites in
callus culture
11 1945 LOO Cultures from
stem tipos.
12 1946 BALL First meristem Apical shoot of
culture Lupinus albus and
Tropaeolum majus
12 1952 MOREL & First Dahlia
MARTIN micropropogation
from stem tips,
plants free from
Vrus
13 1954 MUIR First Suspension Nicotiana, Taget.
culture of single
cells and cell
aggregates.
14 1955 MILLER Harmone Kinetine
was discovered
15 1956 ROUTIEN & First US patent for Phascolus
NICKEL the production of
metabolites from
tissue culture
16 1957 SKOOG & Discovered the
MILLER Hormone Auxin:
Cytokinin ratio
regulate the organ
formation
17 1959 TULECKE & First report of Ginkgo, Ilex
NICKEL large scale culture
of plant cells
Kotte mahendar,
M.Pharmacy, (Ph.D), Dept. of Pharmacognosy & Phytochemistry.
Plant tissue culture
18 1960 BERGMANN Development of
plating technique
for isolation of
single cell
19 1965 VASIL & Plant regeneration Nicotiana
HILDEBRAMBT from single cell
cultivated in
hanging droplet.
20 1967 BOURGIN & Invitro production NICOTIANA,
NITSCH of haploid plants Datura
from pollen
culture
21 1970 POWER Successful
Protoplast fusion
22 1970 MAHESWARI & Successful Anther
GUHA culture
23 1971 NAGATA & Regeneration of Niictiana
TAKEBA plants from
cultured
protoplast.
24 1974 REIHARD Biotransformation
in plant tissue
culture
25 1977 NAGUCHI Cultivation of Nicotiana
tobacco cells in
reactors
26 1978 MELCHERS First intergenic Pomato
somatic hybrid
plant from
protoplast fusion
27 1979 BRODELIUS Use of
immobilized plant
cells from
bitransformation
28 1982 TABATA & Large scale Lithospermum
FUJITA production of
Shikonin
29 1984 MITSCUI & First industrial Lithospermum
Petrochemicals production of
Shikonin
30 1989 FERUYA. et al Root culture with Panax root
higher
glycosylation

Basic Requirements and Techniques of Plant Tissue Culture:

Well designed PTC laboratories generally have good work flow allowing the actual movement of
supplies, people, culture and the finished products. Inorder to keep airborn contaminats to a minimum,
the lab has very few entry sites and generally has sufficient areas designed as clean rooms with purified
air flowing (HEPA-filters) under positive pressure.
The main requirements of plant tissue culture are:
Kotte mahendar,
M.Pharmacy, (Ph.D), Dept. of Pharmacognosy & Phytochemistry.
Plant tissue culture
1. Laboratory Organisation
2. Aseptic Conditions
3. Culture Media
Laboratory Organization: In a standard tissue culture lab, there must be a few basic facilities like:
i. A Media Room for preparation, sterilization and storage of culture media.
ii. Facilities for washing of lab-wares, explants, etc.
iii. Space for storage of lab-wares.
iv. Culture rooms or incubators where conditions of temperature, humidity and light etc. can be
maintained.
v. Observation and Data Collection area.
vi. Space for Shipping

Aseptic Conditions: Maintenance of aseptic conditions is the most critical and difficult aspect of in-vitro
culturing experiments. Aseptic condition mean the conditions free from any type of microorganisms (so
as to prevent the loss of experiment by contamination). For this, sterilization (i.e., complete removal or
killing of microbes) is done. The most common contaminants in culture are fungi and bacteria.
Measures to be taken for maintaining asepsis during tissue culture are:
i. Sterilization of the culture vessels using detergents, autoclaves, etc.
ii. Sterilization of instruments like forceps, needles etc. by flame sterilization.
iii. Sterilization of culture medium using filter sterilization or autoclaving methods.
iv. Surface sterilization of explants using surface disinfectants like Silver Nitrate (1%), H 2O2 (10-12%),
Bromine water (1-2%), Sodium Hypochlorite solution (0.3-0.6%), etc.
The whole procedure of plant tissue culture is to be carried out essentially under aseptic conditions. So,
the overall design of the laboratory must focus on the maintenance of aseptic conditions. Secondly, the
worker is also required to have proper knowledge of operating various equipment’s like pH meter,
balance, laminar air flow, microscope, etc. While performing the tissue culture experiments there must
present the first aid kits and fire extinguishers in the laboratory to avoid any mishap or accident. In
addition, proper attention should be given while handling the toxic chemicals and all the chemicals should
be kept in correct labeled containers and bottles.

Culture Media:
The formulation or the medium on which the explants is cultured is called culture medium. It is composed
of various nutrients required for proper culturing. Different types of plants and organs need different
compositions of culture media. A number of media have been devised for specific tissues and organs.
Some important of them are:
LS (Linsmaier and Skoog) Medium
MS (Murashige and Skoog) Medium is used to iduce organogenesis and regeneration of plant in cultured
tissues.
B5 (Gamborg’s) Medium is originally designed for cell suspension culture and callus culture, now it is
using for Protoplast culture with some modifications in media composition.
White’s Medium is one of earliest medium developed for the root culture.
N6 medium (Chu) is used for cearl anther culture besides other tissue culture.
Nitsch’s medium (Nitsch and Nitsch) is used for anther culture.
Synthetic media
Natural media

Important constituents of a culture medium are:

Many elements are needed for plant nutrion and their Physiological functions. These have to be supplied
in the cultured medium to support adequqte growth of cultures invitro.
1. Organic suppliments

Kotte mahendar,
M.Pharmacy, (Ph.D), Dept. of Pharmacognosy & Phytochemistry.
Plant tissue culture
2. Inorganic suppliments
3. Carbon and energy source
4. Growth harmones
5. Gelling / Solidifying agents
6. PH of the medium

(i). Organic supplements:

(a) Vitamins like thiamine (B1), Pyridoxin (B6), Nicotinic Acid (B3), etc.
(b) Antibiotics like Streptomycin, Kanamycin;
(c) Amino Acids like Arginine, Asparagine.

(ii) Inorganic Nutrients:

Micronutrients as Iron (Fe), Manganese (Mn), Zinc (Zn), Molybdenum (Mo), Copper (Cu), Boron (B).
Macronutrients include six major elements as Nitrogen (N), Sulphur (S), Phosphorus (P), Potassium (K),
Calcium (Ca), Magnesium (Mg).
(iii) Carbon and Energy Source:
Most preferred carbon source is Sucrose. Others include lactose, maltose, galactose, raffinose, cellobiose,
etc.
(iv) Growth Hormones: the growth and the development of higher plant tissue invitro is controlled by
endogenous plant growth harmones. They promote growth, differentiation and organogenesis of plant
tissue in cultures.
a. Auxins-mainly for inducing cell division, cell elongation and formation of callus in culture. At low
concentration it promotes root culture while high concentration promotes Callus formation. Auxins are
IAA,IBA,NAA,NOA,2,4-D, 2,4,5-T. among the all Auxins 2,4-dichlorophenoxy acetic acid(2,4-D) is
most effective and is widely used in culture media.
b. Cytokinins-mainly for modifying apical dominance and shoot differentiation. Chemically these are
derivatives of Purine (Adenine). Involved in cell division and somatic embryogenesis. It promote RNA
synthesis, stimulates protein and enzyme activities in tissues. These are Zeatine, Kinetin, BA,BAP and
DPU. Among the above all Kinetin and BAP are frequently used in culture media.
c. Abscisic Acid (ABA)-Used occasionally. The callus growth cultures may be stimulated or inhibited by
ABA. This is largely depends up on the nature of the plant species.ABA is an important growth regulator
for induction of embryogenesis.
d. Gibberellins-Used occasionally about 20 gibberellins are identified as growth regulators.GA 3 is most
commonly used for tissue culture. GA3 promotes growth of cultured cells, enhances the callus growth and
induces Dwarf plantlets to Elongate. These are capable of promoting and inhibiting growth of tissue
culture depending on the plant species. They usually inhibits adventitious and shoot formation.
e. Ethylene- plays an important role in somatic embryogenesis, morphogenesis and regeneration from
invitro cultures. Stimulate the production of Ethylene gas.
Gelling / Solidifying Agents: These are added to media to make them semisolid or solid. Agar, Gelatin,
Alginate etc. are common solidifying or gelling agents.
Other Organic Extracts:
Sometimes culture media are supplemented with some organic extracts also like coconut milk, orange
juice, tomato juice, potato extract, etc.

Kotte mahendar,
M.Pharmacy, (Ph.D), Dept. of Pharmacognosy & Phytochemistry.