Tissue Culture

Types of Tissue Culture

The types of tissue culture can be grouped by
the structures formed in culture.
Plantlets
Seedlings
Callus
Somatic Embryogenesis

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve

Tissue Culture
Plantlet formation
1. Axillary shoot formation
Meristem culture

Shoot culture
2. Adventitious shoot formation
Diploid plant regeneration
Haploid and triploid regeneration

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve

Tissue Culture
Axillary shoots form directly from existing meristems.
The initial explant is a shoot tip or stem pieces.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve

Tissue Culture
Adventitious shoots form indirectly after the initiation of new
meristems. The initial explant is an internodal stem piece, leaf,
root or other tissue without a pre-existing meristem.
Indirect organogenesis

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve

Tissue Culture
Indirect adventitious shoots may also form after an intermediate
callus step.
Indirect organogenesis

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve

Tissue Culture
Plantlet formation - Meristem cultures

Uses a shoot tip that is less than 1 mm

in size.
Can be used for micropropagation.

Mostly used for virus elimination.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve

Tissue Culture
Plantlet formation - Shoot cultures

This is the most common

form of micropropagation.
Uses a portion of the stem
with one to several nodes.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve

Tissue Culture
Plantlet formation - Shoot cultures

1. Axillary branching

4. Pseudocorms

2. Nodal cultures

5. Minitubers

3. Stool shoots

6. Micrografting

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve

Tissue Culture
Plantlet formation - Shoot cultures
Axillary branching
The initial explant contains
several nodes on a single stem.

Lateral shoots are

stimulated to form a dense
clump of shoots.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve

Tissue Culture
Plantlet formation - Shoot cultures

Axillary branching

The clump is separated

into smaller clumps during
subculturing.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 10

Tissue Culture
Plantlet formation - Shoot cultures
Nodal cultures
Used with species with strong
apical dominance.
Long shoots are cut into single
nodes and placed vertically in
the medium.
The new shoot elongates and
the process is repeated.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 11

Tissue Culture
Plantlet formation - Shoot cultures
Stool shoots
A shoot with several nodes is
layered horizontally on the
medium.

A thicket of stems emerge.

Clumps are separated and
subcultured.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 12

Tissue Culture
Plantlet formation - Shoot cultures

Pseudocorms
Pseudocorms are the
structures initiated after seed
germination in orchids.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 13

Tissue Culture
Plantlet formation - Shoot cultures
Pseudocorms
Orchids in culture can continue to
produce clusters of pseudocorms.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 14

Tissue Culture
Plantlet formation - Shoot cultures

Minitubers
Minitubers can be produced in
plants that naturally form
tubers like potato and yam.
Each minituber can be used as
a propagule.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 15

Tissue Culture
Plantlet formation - Shoot cultures
Micrografting - an alternative to meristem culture

Production of disease-free plants.

Virus indexing.

Micropropagation.
Rejuvenation by serial micrografting.
Send germplasm between countries.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 16

Tissue Culture
Plantlet formation
1. Axillary shoot formation
Meristem culture

Shoot culture
2. Adventitious shoot formation
Diploid plant regeneration
Haploid and triploid regeneration

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 17

Tissue Culture
Plantlet formation - Adventitious shoots

Diploid plant regeneration

Explants include:
Leaf pieces

Leaf disc

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 18

Tissue Culture
Plantlet formation - Adventitious shoots

Diploid plant regeneration

Explants include:
Leaf pieces, petioles

Petioles

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 19

Tissue Culture
Plantlet formation - Adventitious shoots

Diploid plant regeneration

Explants include:
Leaf pieces, petioles, bulb scales
Bulb scales

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 20

Tissue Culture
Plantlet formation - Adventitious shoots

Diploid plant regeneration

Stem internode

Explants include:
Leaf pieces, petioles, bulb scales,
stem internodes

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 21

Tissue Culture
Plantlet formation - Adventitious shoots

Diploid plant regeneration

Explants include:
Leaf pieces, petioles, bulb scales,
stem internodes, roots
Root piece

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 22

Tissue Culture
Plantlet formation - Adventitious shoots

Diploid plant regeneration

Explants include:
Leaf pieces, petioles, bulb scales,
stem internodes, roots and callus.

Callus

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 23

Tissue Culture
Plantlet formation - Adventitious shoots

Diploid plant regeneration

Used for micropropagation,
especially in monocots.

Hosta

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 24

Tissue Culture
Plantlet formation - Adventitious shoots

Diploid plant regeneration

Adventitious shoot
regeneration is a key step in
systems used to transform
(genetically engineer) plants.

Genetic engineering with a

blue marker gene.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 25

Tissue Culture
Plantlet formation - Adventitious shoots
Haploid plant regeneration
Anther culture is a procedure for
obtaining haploid (half the
chromosome number) plants
from normally diploid plants.
The discovery that pollen grains
could develop into embryos was
made by accident with Datura by
Guha and Masheshwari in 1964.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 26

Tissue Culture
Plantlet formation - Adventitious shoots
Haploid plant regeneration
Anther culture is used in plant
breeding to produce haploid
plants to allow crossing between
species with different
chromosome number and to
create dihaploids.

The procedure has been

described for tobacco, Datura,
potato, rape seed, cereals and
grasses.
Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 27

Tissue Culture
Techniques used to regenerate plants

1. Plantlet formation

2. Seedling formation
3. Callus formation
4. Somatic embryo formation

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 28

Tissue Culture
Seedling formation
Seed culture
Embryo culture
Embryo rescue
Ovule and ovary culture

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 29

Tissue Culture
Seedling formation
Seed culture
Common practice in orchids.
Orchid seed is so small and
lacks storage reserves.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 30

Tissue Culture
Seedling formation
Seed culture
In nature, orchid seeds rely
on symbiotic mycorrhizal
fungi associations to
germinate.
However, they can germinate
and develop into seedlings on
the proper tissue culture
medium without the fungal
association.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 31

Tissue Culture
Seedling formation - Embryo culture

Mature embryos germinate

easily in tissue culture to
form embryos.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 32

Tissue Culture
Seedling formation - Embryo culture

Mature embryos germinate

easily in tissue culture to
form embryos.
Used for research, to
create understocks for
micrografting and
sometimes for propagation.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 33

Tissue Culture
Seedling formation - Embryo rescue
Immature embryos are removed from the ovule
before they abort.
Primarily used in breeding to rescue embryos
from interspecific crosses.

These crosses are initially successful and the

embryo forms. However, they never reach
maturity.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 34

Tissue Culture
Seedling formation - Ovule and ovary culture

Also used in plant breeding for difficult crosses.

Unfertilized ovules are excised, placed in tissue
culture and artificially pollinated.

Fertilized ovules develop in culture to form seeds.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 35

Tissue Culture
Techniques used to regenerate plants
1. Plantlet formation
2. Seedling formation
3. Callus formation
4. Somatic embryo formation

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 36

Tissue Culture
Callus formation

Stationary callus cultures

Callus can be derived from any
tissue explant.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 37

Tissue Culture
Callus formation

Stationary callus cultures

Callus can be derived from
any tissue explant.
Used for research, breeding,
genetic transformation and
production of secondary
compounds.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 38

Tissue Culture
Callus formation
Suspension callus cultures

Callus from stationary cultures

can be placed in liquid medium
and shaken to form suspensions.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 39

Tissue Culture
Callus formation
Bioreactors
Bioreactors are automated
systems for production of callus.
Can be used to produce
enzymes, medicines, natural
flavors and colors.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 40

Tissue Culture
Callus formation - Protoplasts

Protoplasts are isolated single

cells from which the cell wall
has been removed by fungal
enzymes.
Used for basic research and
breeding.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 41

Tissue Culture
Callus formation - Protoplasts
Two protoplasts can fuse to
form a single cell. The nuclei
combine and form a single
new genotype.
This is a way to bypass
natural sexual incompatibility.

New cell walls form and

shoots can be regenerated
from the resultant callus.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 42

Tissue Culture
Techniques used to regenerate plants
1. Plantlet formation
2. Seedling formation
3. Callus formation
4. Somatic embryo formation

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 43

Tissue Culture
Somatic embryogenesis

Embryos are induced to

form on vegetative tissue.
They can be used for
clonal propagation or to
recover genetically
transformed plants.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 44

Tissue Culture
Somatic embryogenesis
Somatic embryos go through
similar stages of embryogenesis
compared to zygotic seeds.
However, somatic embryos lack
endosperm, seed coats and
form smaller and often
malformed cotyledons.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 45

Tissue Culture
Somatic embryogenesis
Developmental stages during somatic embryogenesis in interior
spruce (Picea glauca x P. engelmanni ).

Photos provided by Patrick von Aderkas and Lisheng Kong (Univ. of Victoria, Canada).
Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 46

Tissue Culture
Somatic embryogenesis
Stages of somatic embryogenesis.
1. Induction

2. Development.
3. Conversion.
4. Germination.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 47

Tissue Culture
Somatic embryogenesis
The induction stage usually requires 2,4-D.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 48

Tissue Culture
Somatic embryogenesis
The development stage is inhibited by 2,4-D and is
often enhanced by ABA.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 49

Tissue Culture
Somatic embryogenesis
Removal of 2,4-D from the media and the addition of ABA
allows the progression of embryogenesis to the heart and
cotyledon stages of embryogenesis.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 50

Tissue Culture
Somatic embryogenesis
Conversion prepares the embryo for germination and can
be aided by desiccation.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 51

Tissue Culture
Somatic embryogenesis
Germination usually proceeds normally, but gibberellin can
be used to enhance germination.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 52

Tissue Culture
Somatic embryogenesis
After germination, a plantlet
can be formed.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 53

Tissue Culture
Somatic embryogenesis
Direct somatic embryogenesis
Immature zygotic embryos or
embryo parts are placed on a
2,4-D induction medium.

These are moved to a

development medium without
2,4-D but supplemented with
ABA. A gently dehydration step
may help somatic embryos to
germinate.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 54

Tissue Culture
Somatic embryogenesis
Indirect somatic embryogenesis
Seedling tissue is induced to
form polyembryogenic masses
in a liquid culture containing
2,4-D.
Polyembryogenic masses can be
sized using a screen mesh for
uniformity before being placed
on a development medium
without 2,4-D.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 55

Tissue Culture
Somatic embryogenesis - Synthetic seeds

A synthetic seed
contains a somatic
embryo or plantlet
surrounded by an
artificial polymer
seed coat.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 56

Tissue Culture
Somatic embryogenesis - Synthetic seeds
Proposed uses for synthetic
seeds include:
1. Clonal propagation to
replace traditional seeds.

2. To replace hand pollinated

hybrid seeds.
3. Carriers for beneficial
microorganisms, pesticides,
and growth regulators.

Hartmann and Kesters Plant Propagation, Principles and Practices 8th ed. Hudson Hartmann, Dale Kester, Fred Davies, Jr. and Robert Geneve 57