This document outlines the procedure for tissue culture and micropropagation through in vitro culture. It involves 3 major stages: 1) preparation which includes explant selection, sterilization and inoculation onto nutrient medium. 2) Sub-culturing to induce callus formation. 3) Organogenesis where the callus is transferred to auxin-rich medium to induce root formation, followed by hardening in a greenhouse and transplantation to soil. Key steps include explant sterilization using antiseptic agents, preparation of solidified nutrient medium, incubation for callus induction, sub-culturing every 28 days to prevent nutrient depletion, and a final acclimatization and transplantation stage.

1. TISSUE CULTURE/
MICROPROPAGATION/
IN VITRO CULTURE/ IN VITRO
PROPAGATION/CLONAL
PROPAGATION
Dr Sheeja T Tharakan

2. Procedure or Protocol
 3 major stages
 Preparation stage
Preparation of nutrient medium
Explant selection
Explant sterilization
Explant inoculation
 Sub-culturing
 organogenesis
 Direct regeneration
Cultrue transferred to auxin rich medium(roots)
(Green houses)- hardening & acclimatization
Transplantation to soil

3. 1) Explant selection and isolation
 First step
 Ideal plants from actively growing parts
 Shoot tips, axillary buds, leaves (healthy plants)
 Use sterilized blade, razor etc.
 Presene of parenchyma first & foremost
consideration
 Quickly responds to culture
 2 to 5mm in size
Procedure or Protocol

4. 2) Explant sterilization
Using antiseptic agents
initial sterilization
-Detergents, 70% alcohol
Final sterilization
-Chlorine water/ hypochlorite of
Na/Ca(1-2%)/0.1% mercuric chloride
Procedure /Protocol

5. 3)Preparation of nutrient medium
 Appropriate culture medium
 Solidification by agar
 Liquid medium
 Transferred to culture vials under aseptic
conditions
Procedure or Protocol

6. 4)Explant inoculation and incubation
 Inoculation
Transfer of explants to nutrient medium
(culture vials, asceptically)
 Inoculum
Explant introduced to the culture medium
 Incubation
230 to 27oC
Alternate dark & light
Use cool white fluorescent bulbs
High relative humidity uniform air circulation
Procedure or Protocol

7. 5)Callus induction
 Nutrient medium + auxin - induces cell division
 Callus
Surface of the explant covered by a mass of thin walled
and undifferentiated mass parenchymatous cells
 Totipotent
 Callus formation depends on
Nature of explant, composition of the medium
Environmental factors
 Meristematic tissues divide more rapidly
…contd..
Procedure/Protocol

8. 5)Callus induction -3 stages
 Induction
 Cell division
 Cell differentiation
 Callus – 2 types
 Friable callus
 loosely arranged
Easily manipulated for suspension culture
 Compact callus
compactly arranged,
Not ideal for suspension culture
Procedure/Protocol

9. 6)Sub-culturing
 Callus (28 days)
Nutrient depletion
Accumulation of toxic metabolites
Scarcity of water
Death of callus
Procedure/Protocol

10. 7)Somatic embryogenesis
 Formation of somatic embryo from calluses-
embryoids
 Regeneration – somatic buds –germination -plantlet
Procedure/Protocol

11. 8))Organogenesis
 Formation of root and shoot from somatic
embryo/somatic buds
 Begin when stimulation given by chemicals
 Caulogenesis – shoot formation
 Rhizogenesis -Root initiation
 Controlled by hormones
 High auxin:cytokinin-induce roots
 Low ratio – induce shoots
Procedure/Protocol

12. 8))Organogenesis
 Torrey’s hypothesis of organogenesis
 Meristemoids- initite formation of primordium
 Depend on facors shoot/root/embryoid
 Auxin:cytokinin ratio- (1:100)- callus
 Cytokinin:Auxin (1:100) -buds
Procedure/Protocol

13. 9)Direct regeneration
 direct regeneration is advantageous
Callus causes undesirable variations- somaclonal
variation
Procedure/Protocol

14. Procedure/protocol
10)Acclimatization and transplantation /transfer to the field
 Taken out from the medium
 Remove agar by running water
 Low mineral salt medium (LMSM) for 24-48 hrs
 Transferred to pots (autoclaved- sterilized clay, sand and leaf mould
1:1:1)
 Pots covered with transparent polythene to maintain humidity
 Undisturbed for 15-30 days
 Acclimatized
 Transferred to field