Inoculation of explants

1. To execute inoculation of explants for clonal propagation
BIOTECHNOLOGY OF HORTICULTURAL CROPS
HRT 552

2. A) Explant
Explant is a tissue taken from mother plant,
cultured under aseptic condition on a defined medium
by manipulation of medium component and other
physiological parameters. The nature of the explants to
be used for in vitro propagation is, to a certain extent,
governed by the method of shoot multiplication to be
adopted.

3. Explant
• The tissue obtained from a plant to be cultured is called an explant.
• Explants are small pieces of plant parts or tissues.
• Are aseptically cut and used to initiate a culture in a nutrient
medium.
• Explants can be taken from different parts of a plant such as:
• shoots, leaves, stems, flowers, roots, and from many types of mature
cells provided they are able to de-differentiate into totipotent cells.

4. Selection of Explants:
• The correct choice of explant material can have an important effect
on the success of tissue culture. The choice of explants depends on:
1) the kind of culture to be initiated
2) the purpose of the proposed culture
3) the plant species to be used

5. Basic Procedure of Tissue Culture

6. Selection of Explants:
• The correct choice of explant material can have an important effect on
the success of tissue culture. The choice of explants depends on:
1) the kind of culture to be initiated
2) the purpose of the proposed culture
3) the plant species to be used

7. S.No. Explants used Needs
1. Explants with pre-
formed vegetative bud
For enhanced axillary branching.
2. Shoot tips To produce virus-free plant from an infected
individual.
3. Nodal cuttings When the stock plant is virus-tested or,
alternatively, virus eradication is not desired.
4. Runners tips For rhizomatic plants, such as strawberry and Boston
fern.
5. Explants are derived from
root, stem, leaf or nucellus
based on their natural
capacity to form
adventitious buds.
For multiplication through adventitious bud formation,
with or without callusing
6. Leaf-base and scale-
base with small basal
plate.
For monocots.
Table: Type of explants used

9. Continue..
• For successful culture initiation, there is a minimum size for explant
per unit culture volume.
• In practice, minimum inoculation size of explant varies according to
the genotype of the plant being cultured and the cultural conditions.
• For commencing suspension cultures it is commonly about 1-1.5 x
104 cells/ml.

11. Contamination Free
• Plants growing in the external environment are invariably
contaminated with micro-organisms and pests.
• These contaminants are mainly confined to the outer surfaces
of the plant, although, some microbes and viruses can even
grow within the root.

12. Continue….
• Most of the surface microbes do not form tight associations
with the plant tissue.
• They are easily removed from the explant by gently rinsing,
and the remaining are killed by surface sterilization.
• Placing the explant in a 70% ethyl alcohol solution prior to
treatment with another disinfectant solution.
• Conducting the sterilization process under vacuum.
• This results in the removal of air bubbles and provides a more
efficient sterilization process.

13. STERILIZATION OF EXPANTS
Sodium
hypochlorite
Calcium
hypochlorite

14. EXPONENTIAL GROWTH

15. •When the cells in culture are reaching at stationary
state it should be sub-cultured.
•Sub-culturing may be in similar or dissimilar type of
media with respect to the requirements.