Plant tissue culture involves growing plant cells, tissues or organs under sterile conditions on a nutrient medium. It has a history dating back to the 1930s and was pioneered by scientists like Haberland and White. Techniques include using explants from plants, sterilizing them, and culturing them on nutrient media containing salts, sugars and growth regulators. This allows for dedifferentiation of cells and regeneration of whole plants. Micropropagation specifically refers to rapidly multiplying shoots in culture. It has five stages - selection of stock plants, initiation of culture, shoot multiplication, rooting, and hardening. It has advantages like producing many pathogen-free plants quickly from small explants.

1. PLANT TISSUE CULTURE
Prepared by
Dr. K. Vanangamudi
Formerly Dean (Agriculture), AC & RI, Coimbatore,
Dean, Adhiparashakthi Agricultural College,Kalavai,
Professor and Head - Seed Science and Technology,
Tamil Nadu Agricultural University, Coimbatore.
Plant tissue culture
 Collection of techniques used to maintain or grow plant cells, tissues or organs under
sterile conditions on a nutrient culture medium of known composition.
History of plant tissue culture
Scientists Contribution
Schleiden and Schwann (1938
and 1939)
Cell theory - Cell is the functional unit of living organisms.
Worked in Tradeschantia with knop’s solution.
Habertland (1902) First attempt of plant tissue culture ; Father of tissue
culture
White (1934) First reported for the successful continuous cultures of
tomato root tips in liquid culture and obtained indefinite
growth.
Morel and Martin (1952) Use of meristem tip culture to obtain virus free Dahlias
Gautheret (1959) First hand book on plant tissue culture
Murashige and Skoog (1962) Development of Murashige and Skoog (MS) medium
Guha and Maheshwari (1962) Development of haploids through anther and pollen culture
for the first time.
Power et al., (1970) First protoplast fusion
Larkin and Scowcroft (1981) Introduction of the term somaclonal variation.
Terms and terminology
 Totipotency: Regeneration capacity of plant cell develops into the whole plant.
 Callus: A mass of unorganized, regenerated cells in the culture medium
 Dedifferentiation: Conversion of mature cells in to meristematic state leading to callus
formation.
 Re-differentiation: Unorganized cells become specialized in form (root and shoot)
function as a whole plant.
 Protoplasm (Naked cells): Cell without cell wall

2. Techniques of plant tissue culture
1. Explants
 Plant tissue or organ (Eg. Anther) excised and used for in vitro culture
2. Surface sterilization
 Explant surface should be sterilized to eliminate bacteria , fungi
 1-2% solution of sodium or calcium hypochlorite or 0.1% solution mercury chloride.
 Rinse the explant in sterilized distilled water to remove disinfectant.
 Then, sterilize explants under aseptic conditions i.e., laminar flow chamber.
3. Sterilization
 Microbes present in culture media, culture vessels, and instrument are inactivated by
suitable treatment.
o Flame: Forceps, scalpel and needles are sterilized with 95% alcohol and flamed.
o Dry heat: Test tube and culture flasks are heated on a burner
o Ethanol (70%): To sterilize laminar air flow chamber, culture vessels and
hands of workers
o Autoclaving: It is done to sterilize culture media, culture vessel at 1210C and
15 psi for 15-20 minutes.
o Air filter: Air blowing the laminar tools is sterilized by HEPA filter (High-
efficiency particulate absorbing filter and high-efficiency particulate arrestance
filter).
o Filter: Thermobiable constituents (A substance which is subject to destruction,
decomposition, or change in response to heat) like ABA, GA3 and enzymes are
filtered using 0.45µm pore size.
4. Nutrient medium/culture medium
 Plant cell and organs are cultured
 Contains growth regulators, inorganic salts, vitamins, carbon sources (sucrose)
 Growth regulators viz, auxin (Root formation) and cytokinin (Shoot formation)
 Auxin: 2,4 D (0.5-2.0 mg/l), NAA and IAA (Natural), kinetin and benzyl amino purine
(BAP)
o Commonly used kinetin is zeatin.
 Organic supplements: Coconut water, casein hydrolysate and yeast extract
 pH of medium: 5.5 by using1N KOH or HCl.

3.  Solidify agent: Agar (6 g/l)
o Commonly used media (MS medium).
 Callus culture: Cells on the agar medium develop into an unorganized mass callus.
 Suspension culture: In liquid medium, a suspension of free cells and small cell masses.
5. Environmental conditions
 Organ and cell culture is evaluated under controlled temperature, light and RH.
o Temperature: 18-250C
o Light is not essential, but beneficial for plantlet regeneration
 Culture room/ incubator is the best.
6. Sub-culturing
 After a period of time, it is necessary to transfer organs and tissue to fresh media.
 A portion of tissue is used to inoculate in new culture tubes
 Subculture of callus culture is to be done by every 4-6 weeks
7. Plant regeneration and transfer to soil
 Production of roots and shoots from cell and tissue culture - Organ regeneration or
organogenesis. Eg. Colacassia, potato, pea
 Transfer pretreated whole plants from test tube to small pots with soil and cover with
vessels to make them hardy.
 After 3-4 days, covers are removed and kept in diffuse light for 5-10 days.
 Harden the plantlets in a large scale mist chamber.
 Then, plants are transferred into greenhouse and after 1-2 weeks planted in soil and kept
in sunlight
Stages of micro propagation
 Murashige proposed three (I to III) stages
 Debergh and Maene added stage '0'.
 Currently, there are accepted five stages (0 to IV) (Fig. 1).
1. Stage 0: Selection and maintenance of stock plants for culture initiation
 Stock plant are grown under more hygienic conditions to reduce the risk of
contamination.
2. Stage I: Initiation and establishment of aseptic culture
 Explant isolation: Vegetative parts (Shoot tip, meristem, leaves, stems, roots) or
reproductive parts (Anthers, pollen, ovules, embryo, seed and spores).
 Shoot tip and auxiliary buds are most often used.

4.  Size, physiological age, developmental age of explant, and age of stock plant, decide
success rate of stage I.
 Surface sterilization: Ethyl alcohol, bromine water, mercuric chloride, silver nitrate,
sodium hypochlorite, calcium hypochlorite etc. can be used as disinfectant.
 Washing: Washed with water.
 Establishment of explant on appropriate medium: Modifications of Murashige and
Skoog basal medium (Murashige and Skoog, 1962) are most frequently used.
3.Stage II: Multiplication of shoots or somatic embryo formation (Rapid) using a
defined culture medium
 Culture medium
o Inorganics: Macronutrients (N, P, K, Ca, Mg) and micronutrients (B, Co, Cu,
Mn, I, Fe, Zn)
o Organics: Carbon source is needed, since plants do not photosynthesize well in
culture
o Vitamins: Thiamine (essential), myoinisitol, B vitamins, folic acid and biotin
o Growth regulators: Cytokinins, auxins, and GA. ABA rarely used in general.
Cytokinins induce shoot bud formation and auxins induce root formation.
o Complex organics: Natural orange juice, coconut milk and bananas
o Inert supports: Agar, foam rubber, filter paper bridge and liquid
 Rapid multiplication of the regenerative system is carried out for obtaining large
number of shoots.
 About 4.3 x 107 shoots can be produced from a single explant in a year.
 Cultures obtained from stage I are placed on same medium used in stage I, but
cytokinin proportion is increased for stage II to produce numerous shoots.
 Repeated for few cycles until a desired number of shoots are developed to carry out
for rooting.
3. Stage III: Rooting of regenerated shoots or germination of somatic embryos in vitro.
 Shoots from stage II are separated manually from clusters and transferred on a rooting
medium containing an auxin.
 Elongation of shoots prior to rooting, rooting of shoots (Individual or clumps) and pre-
hardening cultures to improve survival are some of the activities.
4. Stage IV: Hardening
 Plantlets removed from plant media are transferred to soil or potting compost

5.  Hardening: When plantlets are taken out of culture, plantlets need time to adjust to
more natural environmental conditions.
 Slowly weaning plantlets from a high humidity, low light and warm environment to
what would be considered a normal growth environment for the species in question.
Fig. 1. Diagrammatic representation of stages of micropropagation
Advantages of micro propagation
 An alternative approach to conventional methods of vegetative propagation.
 A million of shoot tips can be obtained from a small, microscopic piece of plant tissue
within a short period of time and space.
 Shoot multiplication usually has a short cycle (2-6 weeks) and each cycle results in
logarithmic increase in the number of shoots.
 More advantageous in case of bulb or corm producing plants, because mini-tubers or
mini corms for plant multiplication are available throughout the year irrespective of
season.
 Smaller size of propagules is advantageous for storing and transporting as it takes lesser
space.
 Propagules can be maintained in soil free environment which facilitates their storage
on a large scale.

6.  In vitro technique helps to raise pathogen free plant and to maintain them.
 Very useful for dioecious plants, because there the seed progeny yield is 50% male and
50% female.
 Thousands to millions of plantlets can be maintained within the culture vials.
 Through seed production genetically uniform progeny is not possible always; but the
micro propagation method will help to maintain the genetic uniformity in the
propagules.
 Newer tissue material obtained through rDNA technology (Recombinant DNA
Technology) or haploid culture or somatic hybridization can be the source of tissue
material for micro propagation, as it is the easiest method for obtaining the multiple
propagules.
Demerits of micropropagation
 Cost involved in setting up and maintenance of laboratory is very high.
 Tissue culture techniques require skill and manpower.
 Slight infection may damage entire lot of plants.
 Some genetic modification (Mutation) of the plant may develop with some varieties.
 Seedling grown under artificial condition may not survive when placed under
environmental condition.
Commercially propagated plants through micro propagation in India
Category Crops
Fruits Banana, pineapple, strawberry
Vegetables Potato
Spices Turmeric, ginger, vanilla, cardamom
Medicinal plants Aloe vera, geranium, stevia, patchouli, neem
Ornamentals Gerbera, carnation, anthurium, lily, syngonium, cymbidium
Woody plants Teak, bamboo, eucalyptus, populus
Biofuel plants Jatropha, pongamia
Explants and medium used
Species Explant Medium
Gerbera Shoot-tip MS
Rose Shoot tip MS
Carnation Meristem tip MS
Rhododendron Shoot tip Anderson formula
Anthurium Vegetative
buds
MS
Rosemary Shoot tip MS
Banana Suckers MS
Eucalyptus Shoot tip WPM