General procedures for plant tissue culture involve sterilizing glassware and explants, selecting an appropriate culture medium, and incubating the explanted tissue. Explants are sterilized using chemicals like mercuric chloride or sodium hypochlorite. Culture medium is selected based on plant species and experimental objectives, and may contain hormones like auxins or cytokinins. Incubation occurs in sterile conditions at 25°C under light and dark cycles to induce callus growth or organogenesis directly from explants. Acclimatization involves hardening plantlets and transferring them to soil or field conditions.

1. General Procedures Involved in Plant Tissue
Culture
1.Sterilisation of glassware tools/ vessels
Kept overnight in sodium dichromate-sulphuric
acid solution.
Hot air oven -120 deg centigrade ½-1 hour.
2.Preparation and sterilisation of explant
Pretreatment of mother plants
Grown under controlled conditions
Prophylactic spray of fungicides and pesticides.
a) Preparation of explant.
Portion of plant to establish a culture.
Dicot plants- young leaves, petioles, stem and
hypocotyls(seedlings).
Monocots-meristematic regions, leaf bases, young
inflorescences.

2.  b) Surface sterilisation
Excised parts –wash with tap water.
Sterilised with Mercuric chloride(1-2%w/v)
Sodium hypochlorite (0.5-5%w/v) for 15mts.
Waxy layer- pretreated with wetting agents.
Ethanol70-90%, tween 20-/0.05%(polyoxyethylene-
sorbitan-monolaurate)
Explants rinsed thrice in distilled water.
3). Selection of culture medium
Well defined nutrient media
Depends on species and objective of experiment
Dicot tissues-MS media high conc. of
nitrate,potassium and ammonium ions
Suitable portion – removed by knife
Young tissue more suitable.

3. Auxins-IBA and NAA –for rooting
Aux ins and cytokinin for shoot proliferation
2,4-D and 2,4,5-T-effective for callus growth
pH of the medium is adjusted
Poured into culture vessels,plugged and sterilised
4) Incubation of culture orProduction of Callus from
explant
Sterilized explant transferred to defined media
aseptically.
Sterile room ideally ventillated.
Flasks-BOD incubator -25±2ºC(3-8 days)
Light and dark cycles -12 hrs each necessary for
Callus
Undifferentiated amorphous cell mass
(shoot tips, stem, root, leaf, inflorescence)

4.  INDIRECT ORGANOGENESIS
Intervening callus stage is present.
Primary explant→Callus →Meristemoid →Organ
Primordium.
DIRECT ORGANOGENESIS
Without intervening callus phase.
Primary explant→Meristemoid →Organ
Primordium
5)Acclimatizing and propagating plant tissue
culture shoots
Sufficient no of plants survive & grow vigorously
when transferred to soil.

5.  Direct field rooting of the propagules.
 Hardening and lignification of the propagule
prior to root induction.
 Hardened propagules exposed to root induction.
 Removed from the medium prior to root
emergence.
 Plantlet s immediately transferred to field where
root formation occurs.
 Avoid root shock.
 Grown in a greenhouse.

6.  PRESENTED BY
 PRASANTH B
 ASST. PROFESSOR
 NIRMALA COLLEGE OF PHARMACY
 MUVATTUPUZHA