Tissue Culture
Problems encountered in tissue culture
1. Hyperhydricity
2. Internal pathogens
3. Phenolic exudation
4. Shoot-tip necrosis
5. Tissue proliferation
6. Somaclonal variation
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve
1
Tissue Culture
Hyperhydricity
Also called vitrification in older
literature.
Tissue becomes water-soaked and
translucent.
Caused by excessive water uptake.
Usually controlled by changing
agar concentration or source.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve
2
Tissue Culture
Internal pathogens
Can be either virus or
bacteria from the initial
explant.
Bacteria are difficult to
eliminate in some plants.
Antibiotics are used in
some difficult cases.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve
3
Tissue Culture
Phenolic exudation
Phenolic exudates are a
frequently encountered
problem with establishing
explants (especially woody
perennials) in culture.
These phenolic compounds
oxidize to form a brown
material in the medium and
inhibit shoot formation.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve
4
Tissue Culture
Phenolic exudation
Treatments to minimize exudation include antioxidant like
ascorbic acid; absorbents like activated charcoal; or frequent
transfers to new media.
No additive
Ascorbic acid
Activated charcoal
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve
5
Tissue Culture
Shoot tip necrosis
Shoot tip necrosis occurs in
some woody perennial tissue
cultures when actively growing
shoot tips develop tip die-back.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve
6
Tissue Culture
Shoot tip necrosis
Shoot tip necrosis occurs in
some woody perennial tissue
cultures when actively growing
shoot tips develop tip die-back.
This condition is usually
caused by a calcium deficiency
in the medium.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve
7
Tissue Culture
Habituation
Habituation is when a culture
continues to develop in the
absence of auxin or cytokinin.
For example, shoot cultures
habituated for cytokinin
would continue to produce
new shoots on a cytokininfree medium.
Habituated pawpaw cultures
continue to produce shoot bud
clusters on cytokinin-free medium.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve
8
Tissue Culture
Tissue proliferation
Tissue proliferation is the formation of a gall-like
protuberance on some micropropagated plants especially
ericaceous plants.
It is most likely caused in sensitive plants by in vitro
conditions and not a pathogen or genetic mutation.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve
9
Tissue Culture
Tissue proliferation
Tissue proliferation may be the
expression of naturally occurring
growths in plants like
Rhododendron induced by the
tissue culture environment.
Naturally occurring basal
growths in Rhododendron.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 10
Tissue Culture
Variation in micropropagated plants
1. Genetic or chimeral variation.
2. Transient phenotypic variation.
3. Epigenetic (rejuvenation).
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 11
Tissue Culture
Somaclonal variation
Somaclonal variation occurs because of genetic mutation
caused by in vitro conditions or by chimeral separation.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 12
Tissue Culture
Somaclonal variation
Somaclonal variation is usually undesirable. Typical types of
mutations include changes in plant form.
Chimeral
variegation.
Change in leaf size.
Various forms of
fasciation.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 13
Tissue Culture
Somaclonal variation
In some cases, somaclonal variation can lead to new
cultivars that may have desirable ornamental
characteristics or increased pest resistance.
New leave patterns developed in
foliage plant cultures.
Poplar cultures being screened for
induced disease resistance.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 14
Tissue Culture
Somaclonal variation
The occurrence of somaclonal
variation can be reduced by:
1. Avoid long term cultures.
2. Use axillary shoot induction
systems where possible.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 15
Tissue Culture
Somaclonal variation
The occurrence of somaclonal
variation can be reduced by:
1. Avoid long term cultures.
2. Use axillary shoot induction
systems where possible.
3. Propagate chimeras by other
clonal systems.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 16
Tissue Culture
Transient phenotypic variation
1. Increased vigor.
2. Changes in developmental stage.
3. Increased axillary branching.
Increased basal branching in Rhododendron and Hydrangea.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 17
Tissue Culture
Epigenetic variation
1. Rejuvenation.
2. Flowering.
Micropropagated eastern
redbud plants.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 18
Tissue Culture
Epigenetic variation
Cuttings that fail to root from
mature stock plants often can
be rooting from microcuttings
from tissue culture.
This rejuvenation (also called
invigoration) can carryover to
new plants for a short time.
Rooted microcuttings in
eastern redbud.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 19
Tissue Culture
Epigenetic variation
Cuttings from birch before and after Micropropagation
show a typical rejuvenation / invigoration.
Cutting type
Rooting %
Seedling
87
Mature
31
Microcutting
95
Next generation
75
Struve and Lineberger, 1988.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 20
Tissue Culture
Epigenetic variation
Most red maple propagation is based on taking cuttings from
stock plants rejuvenated / invigoration through Micropropagation.
Two cutting crops can usually be taken from the stock plants
before the rooting potential is lost.
Hartmann and Kester’s Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 21