• Recalcitrance reffers to inability of
plant tissue culture to respond to
culture manipulation.(loss of
morphogenetic competence and
• Free radicals have an important role
in the metabolism and development
of aerobic organisms.
4. • free radical-mediated stress has a role in tissue
• In vitro plant systems have shown that tissue
cultures produce free radicals, lipid peroxides
and toxic, aldehydic lipid peroxidation products.
• antioxidant protection is compromised oxidative
stress ensues and free radicals and their
reaction products react with macromolecules
such as DNA, proteins and enzymes, causing
cellular dysfuction and, as a result, the cultures
5. • All aerobic organisms are totally dependent
upon redox reactions and the transfer of single
electrons and many life processes involve free
• Free radicals and their reaction products have
an important role in plant stress (Hendry and
Crawford, 1994) and senescence.
6. How to overcome ?
• Juvenile tissue can be selected as explant
• Parts of the desired plant rejuvinated by
treatments like cytokinine spray on selected
• Bacteria, fungi, moulds and yeasts are
common contaminating microorganisms
in tissue culture.
• In plants they occur as microflora
associated with roots (rhizosphere and
rhizoplane) leaves (phyllosphere and
phylloplane), other plant parts and
subliminally as endophytes in plant
tissues and vegetative propagules.
8. • Contamination in tissue culture can
originate from two sources, either
through carry over of
microorganisms on the surface or
in the tissues of explants, or
through faulty procedures in the
• Many of the microorganisms that
are likely to be present intercellular,
in plant tissues will be capable of
growth on the plant tissue culture
medium, although some may be
inhibited by the high salt or sucrose
concentration and the pH (Cassells et
9. How to overcome?
• Wear gloves and a lab coat and keep long hair
• Work in a laminar flow hood when passaging
• Wipe down working surfaces with ethanol.
• Use sterile equipment.
10. • Stay as organized as possible—label everything
and set up all of your materials before getting
• Inspect all equipment and media for visible
contamination before use.
• If you must completely remove a lid from a tube,
plate or bottle, set it down within the hood with
the open surface facing up. Otherwise, keep
tubes, plates or bottles closed as much as
11. • Do not pass your hands/arms over any open
bottle, plate or tube.
• Use proper antibiotics in your culture media.
• When finished, dispose of materials properly,
wipe down working surfaces with ethanol, and
turn on UV lamp within laminar flow hood for 10
minutes to sterilize the area.
14. 3.Phenolic Browning
• Many plants are naturally rich in polyphenolic
compounds that are commonly regarded as
inhibitory agents. In most of the cases, when
these plants are cultured in vitro, the culture
medium turns brown.
• Phenolic browning caused by the accumulation
and oxidation of phenolic compounds.
• Addition of antioxidants to medium was more
effective to reduce the browning.
15. • preventative approach is inhibiting the activity of
the phenylalanine ammonia lyase enzyme
(PAL), thereby reducing the biosynthesis of
16. How to overcome?
• Culture Bottles are kept in dark condition
• Activated charcoal used in media
• Use of antioxidant mixture( Ascorbic acid
0.1gm/ltr+ Citric acid0.15gm/ltr)
• Polyvinylpyrrolidone, PVP-40 acts as a
19. 4. Seasonal Variation
• In Invitro culture seasonal variation is also
effected such as relative humidity, Dry season.
• When too dry nutrient medium evaporates
• In extream moist climate such as poor tropical
region fungi is effected on media
• Dust in air is also a major source of bacterial
• Small insects insects that enter incubation room
through undetermined avenues is also effects
the culture tube.
21. 4. Vitrification
• Hyperhydricity is the physiological malformation
due to excessive hydration, low lignification and
reduced mechanical strength of tissue culture
• hyperhydricity in plant tissue cultures are those
factors triggering oxidative stresses such as
high salt concentration, high relative humidity,
low light intensity, gas accumulation in the
atmosphere of the jar, length of time intervals
22. • High ammonium concentration also contributes
• The reasons include low calcium content in
culture medium, gas builtup within the
container, High concentrations of salts in
culture medium, low light intensity among
• Vetrification is also seen in culture bottles kept
in same container.
25. 5. Somaclonal Variation
• Phenotypic change
• It Occurs due to change in chromosome
number and/or structure – Genetic
• Most of the somaclonal variations are not
• Variations are unpredictabled and uncontrolled
• They are not stable and heritable
• Cultiver dependent
26. • Many cell lines cannot exhibit regeneration
Overcome of Somaclonal Variation
• Avoiding long term term cultures
• Axillary shoot induction systems
• Regularly reinitiating clones from new explants.
• Prevent usage of 2-4D IN media
• Chaudhury, A. M., S. Letham, S. Craig and E.
S. Dennis (1993). Amp1-a mutant with high
cytokinin levels and altered embryonic pattern,
faster vegetative growth, constitutive
photomorphogenesis and precocious flowering.
Plant J. 4:907-916.
• Lin FL, Sperle K, Sternerg N, Model for
• homologus recombination during transfer of
DNA into mouse L cells: ends in the
recombination process plant biotechnology