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Tissueculture 3 171212101129
Tissueculture 3 171212101129
Lecture 3
By
Dr. Ahmed Metwaly
Objectives:
Explant: choosing:
■ Meristematic tissues, located either on terminal or axillary buds, are
induced to proliferate in response to hormonal treatments.
■ If the objective is to produce virus-free plants from an infected
individual it becomes necessary to start with sub-millimeter shoot tips
and away from the infection.
■ If the objective is an enhanced auxiliary branching, only the explants
which carry a pre-formed vegetative bud will be suitable.
■ Explant Sterilization
■ In this stage an explant is surface sterilized and transferred into
nutrient medium.
■ Generally, the combined application of bactericide and fungicide
products is suggested. The selection of products depends on the
type of explant to be introduced.
■ The surface sterilization of explant in chemical solutions is an
important step to remove contaminants with minimal damage to
plant cells .
■ The most commonly used disinfectants are sodiumhypochlorite,
calcium hypochlorite , ethanol and mercuric chloride (HgCl2).
■ The cultures are incubated in growth chamber either under light or
dark conditions according to the method of propagation.
Stage II: Multiplication stage
1. Isolation of protoplast
■ Advantages:
■ Used for variety of tissues and organs such as fruits, roots, petioles,
leaves…
■ Osmotic shrinkage is minimum
■ Cells remain intact and not injured
■ High yield of protoplast
■ Easy to perform
■ More protoplast viability .
Leaf sterilization, removal of
epidermis
Isolated Protoplasm
Procedure
■ Incubation of leaf segments overnight in enzyme solution at
pH 4.5-6.0 & temperature 25-30 0C .
■ Mixture is filtered and centrifuged
■ Protoplast forms pellet
■ Then washed with sorbitol and re-centrifuged
■ Clean protoplasts float
■ They are pipetted out
Purification of protoplast
● Protoplasts are purified by removing:
○ Undigested material (debris)
○ Bursts protoplasts
○ Enzymes
● Debris are removed by filtering the preparation through a nylon
mesh
● Enzymes are removed by centrifugation whereby the protoplasts
settle to the bottom of the tube and the supernatant removed with
the help of a pipette
● Intact protoplasts are separated from broken protoplasts through
centrifugation and removed by a pipette as they are collected at
the top of tube
Purification of protoplast
Protoplast Fusion (Somatic
hybridization)
. The nuclei of two protoplasts may or may not fuse together even after
fusion of cytoplasms.
•When nuclei of two different sources are fused the cells are known as
hybrid.
•Only cytoplasms fuse and genetic information from one of the two
•Regeneration
Plants are induced to regenerate from hybrid calli.
These hybrid plants must be at least partially fertile, in addition to
having some useful property, to be of any use in breeding schemes.
Protoplast Culture
● Isolated protoplast can be cultured in an appropriate
medium to reform cell wall and generate callus
1. Gene Transfer
2. Biological examinations
● Study of Osmotic behavior
● Study of Plasma lemma
● Study of Cell wall formation
● Organelle isolation
● Study of Morphogenesis
● Virus uptake and replication
● Study of photosynthesis
Factors affecting protoplast culture
1. Plant species and varieties
Small genetic difference leads to varying protoplast responses to
culture conditions
2. Plant age and organ
Age of donor plant and its developmental stage (smaller better)
3. Pre-culture conditions
Climatic factors affect the yield of protoplast and response when
cultured
4. Pre-treatment to the tissue,before isolating protoplasts
Cold treatment, plasmolysis and hormone increases the chance of
recovery of viable protoplasts and their plating efficiency
Application of Protoplast