Plant tissue culture provides a means to produce pathogen-free plants by eliminating contaminating bacteria. Bacteria that contaminate plant tissue cultures can originate from the explant tissue, the lab environment, operators, or ineffective sterilization techniques. Contaminants are identified and characterized using biochemical tests and purification methods to determine the source. Producing aseptic cultures involves indexing explants for contaminants, identifying and eliminating the contaminants, and maintaining pathogen-free plant stocks through tissue culture.

1. Plant resistance against
bacteria

2. Importance of controlling contamination
• Medium contains many different bacterial nutrients, both original
constituents of medium and exudates from plant cells.
• When microbe come in contact with plant tissue or medium then
they become contaminated.
• Plant tissue or cells growing in vitro considered to be under some
stress condition and may be predisposed to direct infection even by
bacteria.

3. Contaminating bacteria can be
• Epiphytes
Common
Disinfection can be enough
• Endophytes
More problemetic
Disinfection is not enough
Antibiotics are needed

4. Contaminated Bacteria may originate from
• Explants
• Lab environment

5. • Operators
• Mites and thrips

6. • Ineffective sterilization techniques

7. Procedure for producing aseptic cultures
• Indexing explants and cultures for contaminants
• Identifying source
• Identifying and characterizing contaminants
• Eliminating contaminants

8. Indexing cultures
• Stem slices inoculated into liquid and agar solidified different bacteria
medium.
• Incubated for 3 weeks at 30 c detected most contaminants from more
than 60 aquatic,marsh plant species.
• Usually contaminants grow on 2 of 3 media.

9. Characterization and identification
• Purification by biological methods
• Characterization by biochemical test ,gram staining
• Identification by traditional tests

10. Tissue culture and pathogen free plants
• Another purpose for which plant tissue culture is uniquely suited is in
the obtaining, maintaining, and mass propagating of specific
pathogen-free plants.
• The concept behind indexing plants free of pests is closely allied to
the concept of using tissue culture as a selection system.
• Plant tissues known to be free of the pathogen under consideration
(viral, bacterial, or fungal) are physically selected as the explant for
tissue culture.
.

11. • In most cases, the apical domes of rapidly elongating shoot tips are
chosen.
• These are allowed to enlarge and proliferate under the sterile
conditions of in vitro culture with the resulting plantlets tested for
presence of the pathogen (a procedure called indexing).
• Cultures which reveal the presence of the pathogen are destroyed,
while those which are indexed free of pathogen are maintained as a
stock of pathogen-free material. Procedures similar to these have
been used successfully to obtain virus-free plants of a number of
species and bacteria-free plants of species known to have certain leaf
spot diseases

12. • . The impact of obtaining pathogen-free nursery stock can only be
speculative, since little research documenting viral, bacterial, or
fungal diseases transmitted through propagation of woody
ornamentals is available.