Selection of plant species Selection of mother plant for collection of explant Explant Surface sterilization Media composition for various objectives (Callus, somatic embryo, shoot etc) Genetic fidelity Acclimatization (Primary hardening, secondary hardening, field transfer)

1. By – Dr. Mafatlal M. Kher
Plant tissue culture step by step guide
Date & Time : Wednesday, 08 September 2021
Semester : V
Program : B.Sc. Biotechnology
School : School of Science
Subject code : BSBO502 (Unit I)
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2. Plant tissue culture step by step Guide
 Selection of plant species
 Selection of mother plant for collection of explant
 Explant
 Surface sterilization
 Media composition for various objectives (Callus, somatic embryo, shoot etc)
 Genetic fidelity
 Acclimatization (Primary hardening, secondary hardening, field transfer)
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3. Selection of Plant Species
 Commercial
 Medicinal
 Horticulturally important plant
 Agricultural
 Biodiversity related: Rare, IUCN Red Data List.
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4. Selection of mother/stock plant of explant collection
 Healthy
 Disease free
 Actively growing shoots
 Availability of explant
 High Yielding
 If medicinal: amount of bioactive compounds/gm of fresh or dry weight
 Horticulture: Fruit (quality and quantity)
 Conservation: If plant is rare or not
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5. Explant
Type
 Explant with predetermined meristem: Shoot tip (shoot apical meristem), Node (axillary
meristem).
 Without predetermined meristem: Leaf, internode, petiole, petal, anther, pollen grains,
unfertilized or fertilized ovule.
Size
 Explant size 1.5 to 2.5 cm length for node or minimum one axillary bud ( for nodal explant
in a tree species) or with 2-3 axillary buds in case of herbaceous plants like mint, brahmi
etc.
 Shoot tip culture < 0.2 cm
Orientation
 Node: Upright position (natural position), Leaf natural position lower surface must be in
contact with media, therefore slants are prepared for leaf or explants without
predetermined meristem, sometimes such explants can cultured in petridishesh too. 5

6. Discussion
 Q: If I have inoculated 100 shoot tip and 100 nodes on
shoot culture medium: Which one will perform better? And
Why?
 Answer: Node, because it is having more vigour index (more
surface area, more cell, more cytoplasm).
 Q: Why Shoot tip culture is still in practice?
 Answer: Shoot tip culture is utilized for the production of
Virus free plant.
 What is special in shoot tip ?
• Active cell division, it will not allow virus to replicate.
• Vascular bundles are abs, as virus transmitted via vascular
bundle
• High cell metabolic activity, that will kill virus articles during
cell division.
• Therefore, shoot tip culture is preferred for the production of
virus free plants.
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7. Surface sterilization of explants
Types of surface sterilizing agents and their concentrations, combinations and
duration of the treatment.
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Sterilizing agent Concentration used (%) Duration (min) Effectiveness
Calcium hypochlorite 9-10 5-30 Very good
Sodium hypochlorite 20% v/v commercial solution 5-30 Very good
Hydrogen peroxide 10-12 5-15 Good
Bromine water 1-2 2-10 Very good
Silver nitrate 1 5-30 Good
Mercuric chloride 0.1-1 2-10 Very good
Ethyl alcohol 70 30 sec to 2 Good
Isopropyl alcohol 70 30 sec to 2 Good
Bavistin (50% WP carbendazim.
Carbendazim 50%WP)
2% 2-10 Good (Antifungal)

8. Basic steps for:-
 Callus culture
 Somatic embryogenesis
 Axillary shoot or shoot tip culture
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9. Media composition
Callus culture
1. Callus induction medium (Keep cultures for one to two months)
2. Subculture on shoot induction medium (1-2 months)
3. Multiplication
4. Elongation of shoots
5. Rooting of shoots
6. Genetic fidelity test
7. Primary hardening
8. Secondary hardening
9. Field transfer
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10. Media composition
Somatic embryogenesis
1. Somatic embryo induction medium
2. Maturation of somatic embryo
3. Somatic embryo germination medium
4. Genetic fidelity test
5. Primary hardening
6. Secondary hardening
7. Field transfer
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11. Media composition
Axillary shoot/Shoot tip culture
1. Shoot induction
2. Shoot multiplication
3. Elongation of shoots
4. Rooting of shoots
5. Genetic fidelity test
6. Primary hardening
7. Secondary hardening
8. Field transfer
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