Plant Tissue Culture
Denita Hadziabdic and Neal Stewart
2016
Outline

Introduction and history of tissue culture

Review of anatomy/morphology of plants

Tissue culture advantages and disadvantages

Types of culture and outcomes

Stages of tissue culture

Media preparation

Plant growth regulators
Plant tissue culture
The growth and development of plant seeds,
organs, explants, tissues, cells or protoplasts
on nutrient media under sterile (axenic)
conditions.
Milestones
of plant tissue culture
1904
First attempt to embryo culture of selected crucifers. Hannig B., Bot. Zeitung, 62: 45-80.
1944
First In vitro culture of tobacco used to study adventitious shoot formation.
Skoog F., Am. J. Bot., 31: 19-24.
1949
Culture of fruits in vitro. Nitsch J. P., Science, 110: 499.
1962
Development of MS medium. Murashige T. and Skoog F., Physiol. Plant., 15: 473-497.
1967
Haploid plants from pollen grains of tobacco. Bourgin J. P. and Nitsch J. P., Ann. Physiol.
Veg., 9: 377-382 & 10: 69-81.
1977
Successful integration of transfer DNA (T-DNA) in plants. Chilton M. D. et al., Cell,
11: 263-271.
1985
Infection and transformation of leaf discs with Agrobacterium tumefaciens
and regeneration of transformed plants. Horsch R. B. et al., Science, 227: 1229-1231.
Plant anatomy: why it matters for
tissue culture
Important term:
Explant—the “original” tissues and
cells that are used to start tissue
culture
A
B
Cortex
Epidermis
Endodermis
Vascular
cambium
Phloem
Pericycle
Endodermis
Vascular tissue
Xylem
Cortex
Cross section of a buttercup (dicot)
C
D
Cortex
Epidermis
Xylem
Vascular tissue
Phloem
Pith
Pith
Cortex
Endodermis
Pericycle
Exodermis
Cross section of corn (monocot)
A
B
Vein
Midvein
Epidermis
Buliform cell
Epidermis
Xylem
Mesophyll
Xylem
Phloem
Vein
Phloem
*
Palisade
Mesophyll
Spongy layer
Stoma with
guard cells
Stomata and guard cells
A. Cross section of a dicot leaf.
B. Cross section of a corn (monocot) leaf.
Meristematic tissue
(apical and axillary shoot meristems)
A
B
Stem
Leaves
Leaf petiole
Ax. bud
Leaf petiole
Leaf primordia
Apical meristem
Trichome
Stem
A. Longitudinal section through the
stem tip of bean (Phaseolus vulgaris).
Vascular Tissue
B. Longitudinal section through a
node of catnip (Nepeta cataria).
Flower anatomy
Tissue culture advantages & uses
1. Mass propagation of specific clones
2. Alternative propagation methods (“special needs”
plants)
3. Production of pathogen-free plants
4. Clonal propagation of parental stocks for hybrid seed
production
5. Year-round nursery production
Mass propagation of specific clones
 Reproducing
 superior
copies of an original parent plant
phenotype and genotype
 Controlled
aspects (environment, PGRs) allow
rapid propagation in large numbers
 Rate
can be exponential
 Multiplication
rate of fourfold in cultures subcultured
every 4 weeks= 1,000,000 plants in 10 months
Special needs plants

Useful for :
 plants with slow natural rate of
clonal increase
 high demand and valuable plants;
e.g., orchids (new cultivars, costeffective)
 difficult-to-root plants
 endangered species (conservation)
 plants that cannot be clonally
propagated any other way
http://www.quisqualis.com/tv05tc02p1.html
More tissue culture advantages & uses
6. Genotype modification—targets for transformation
7. Plant regeneration after transformation
8 Germplasm preservation
9. Micrografting
10. Reforestation/preservation
Tissue culture disadvantages

Expensive & sophisticated facilities

Trained personnel/specialized techniques

Contamination can wipe out cultures

Species- and genotype specificity

Production of off-types (variability)
Totipotency or Totipotent:
The capacity of a cell (or a group of cells) to give
rise to an entire organism.
Differentiation:
Transition from meristematic cells to specialized
cellstypically one way path
Explant redefined
X-your-plant: excise tissue from a plant organ and place
it into culture:

The tissue has to be surfacesterilized so it will not have any
contaminating bacteria or fungus

It is then placed inside the tissue
culture vessel (dish, jar, etc.)
containing all required nutrients
and plant growth regulators
Embryogenesis
(or zygotic embryogenesis):
The formation of a zygote after fusion of a sperm and egg cell
and housed in a seed
Natural
Tissue culture outcomes
Somatic embryogenesis
The formation of an embryo from cells other than sperm-egg
fusion; does not occur in nature.
Organogenesis
The development of organs from non-meristematic cells.
How tissue culture is done
Stages of tissue culture
• What is the goal of the experiment?
• Select and prepare explants
• Establishment of aseptic cultures
• Shoot production
• Rooting
M
E
D
I
A
• Transfer plants from cultures and sterile conditions
Laboratory organization
General laboratory and media preparation area
 Transfer area
 Culturing facilities
 Washing facility

Spacious media prep laboratory with “good” water supply and machine-aided media dispensing.
Figure 5.6
Figure 5.6 Researcher working in the
laminar flow hood
Figure 5.7
Figure 5.7 Fungal contamination has taken
over the whole explant.
Figure 5.8 A walk in growth room where
cultures are placed.
Select and prepare explants
 Tissue selection and disinfection
– Pathogen control of stock plants
– Modification of plant physiology
trimming to stimulate lateral shoot growth
 pretreatment sprays containing cytokinins or gibberellic acid
 use of forcing solutions (sucrose and hydroxyquinoline citrate) for
induction of bud break

– Tissue selection (shoot tips, node w/lateral bud, leaf
midrib, bulb scales etc.)
Why?
– Collection prior to flowering
Establishing aseptic cultures

Screening donor plants for suitability

Establishment of sterile culture
 Surface

sterilization prior to placement in culture
Optimal media and vessels
C HOPKNS Ca Fe Mg B Mn Cu Zn Mo Cl
WPM 1 μM BA
MS 1 μM BA
WPM 2 μM BA
MS 2 μM BA
pH needs
to be
~5.8
Figure 5.9
A
B
C
Figure 5.9 Cultures grown in different kinds of vessels –a.
magenta box, b.jar, c. petri plate
Shoot production

Repeated enhanced formation of axillary shoots
from shoot tips or lateral buds

Increased cytokinin concentrations (shoot
proliferation vs. shoot elongation)

Low-to-no auxin

4-8 week subculture intervals (1 cycle)
MS 0 μM BA
MS 2 μM BA
MS 1 μM BA
MS 4 μM BA
Rooting and transfer out of culture

In vitro rooting for few weeks
  cytokinin  auxin

After rooting, transfer to non-sterile environment
(potting mix in pots)

Harden off from 100% humidity to ambient humidity
3 μM IBA
30 μM IBA
100 μM IBA
300 μM IBA
Potential issues when transferring rooted
plants out of tissue culture

Photosynthesis—not needed in
cultures—sugar is provided in media

Dessication from low stomatal control

Low vasculature connectivity

Pests and contaminants
Percentage rooted (%)
Rooted microshoots
100
80
60
40
20
0
0μM IBA
3μM IBA
30μM IBA
100μM IBA
Rooted microshoots
300μM IBA
What’s in tissue culture medium?

Water

Mineral salts

Carbon sources

Vitamins

Plant growth regulators

Other constituents as
desired
Plant growth regulators= hormones

Used in concentrations of
0.001 – 10 µM

Sensitivity to high
temperatures- (IAA,
kinetin, zeatin, etc)

http://www.sivb.org/images/edu/parrot10day.jpg
PGRs interact with specific
target tissues causing
different physiological
responses
http://english.cas.ac.cn/ST/LSB/lsb_progress/201012/t20101221_63355.shtml
Figure 5.2
Figure 5.2 Brassica juncea plants produced from hypocotyls explants. Shoots are
produced when a combination of auxin and cytokinin is used. A. Callus from hypocotyl
explants b. Shoots from callus c. Shoots elongating d. whole plantlets transferred to
soil.
Organogenesis vs somatic
embryogenesis
• Organogenesis: direct production of organs
(stems or roots) from differentiated or
undifferentiated tissue
• Somatic embryogenesis: (brave new world) of
the production of embryos de novo and
without fertilization
http://parrottlab.uga.edu/parrottlab/soyengineering/embryogenesisprotocol.html
Figure 5.10
Figure 5.10 a - Callus tissue, b & c - Shoots arising from
callus (example of organogenesis)
Natural embryogenesis
Figure 5.11
Figure 5.11 Somatic
Embryogenesis: a. Cluster of
somatic embryos b. Globular
embryo c. Embryo becomes
heart shaped as it grows d.
Torpedo shaped embryo is
the next developmental stage
e. The embryo forms
cotyledons as it starts
maturing f. Germinating
embryo
Plant growth regulators (PGRs)

PGRs are signal molecules, present in trace quantities

Differ from animal hormones
 don't
Maintain a condition of
equilibrium/stability
maintain homeostasis
 there are no endocrine organs that produce hormones

When plants respond to stimuli it's controlled by
hormones
Plant growth regulators

Auxin

Cytokinin

Gibberellin

Abscisic acid

Ethylene
Auxins
Compounds capable to induce cell elongation in stems
and otherwise resemble indoleacetic acid in physiological
activity.


The first plant hormones discovered
Most active in young tissues:
 shoot apical meristems
 growing leaves and fruits
Figure 5.3 Structures of
natural and synthetic auxins
used in tissue culture.
Auxins – Functions in TC

Stimulates cell elongation/division

Adventitious root formation (high conc.)

Adventitious shoot formation (low conc.)

Stimulates differentiation of phloem and xylem

Induction of somatic embryos

Callus formation and growth

Inhibition of axillary buds
Cytokinins

Compounds with a structure resembling adenine
which promote cell division and have other similar
functions to kinetin

Concentrations are highest in meristematic regions
and areas of continuous growth potential: roots,
young leaves, developing fruits, and seeds
Figure 5.4 Structures of
natural and synthetic
cytokinins used in tissue
culture.
Effect of different ratio of auxin to cytokinin
http://www.oup.com/uk/orc/bin/0199254680/ch02
Cytokinins – Functions in TC








Adventitious shoot formation
Promotes cell division
Modulates callus initiation and growth
Stimulation of axillary bud breaking and growth
Inhibition of leaf senescence
Kinetin was the first cytokinin discovered
Natural compound but not made in plants - "synthetic"
cytokinin
Zeatin naturally occurring cytokinin in plants - isolated
from corn (Zea mays)
Gibberellins

More than 126 GAs have been identified

Functions in TC:

Stimulates shoot elongation
Stimulates seed germination
Break seed dormancy
Inhibits adventitious root formation



Abscisic acid (ABA)

Naturally occurring compound in plants

Stimulates the closure of stomata (under water stress)

Inhibits cell division:
- Inhibits shoot but not root growth
- Induces seeds to synthesize storage proteins

Promotes dormancy

Induces gene transcription (pathogen defense)

Stimulates the maturation of embryos
Ethylene

Only gaseous PGR - associated with fruit ripening
(used in ancient Egypt to stimulate fig ripening)

Stimulates shoot and root growth as well as
differentiation

Stimulates release of dormancy

Silver nitrate (AgNO3) has anti-ethylene activity

Stimulates flower opening

Promotes flower and leaf senescence

Not commonly used in tissue culture
Hyperhydration (vitrification)
can be caused by ethylene
wikipedia
Figure 5.12 Switchgrass cell suspension types and protoplasts isolation. Scanning
electron micrographs of switchgrass cell suspension types include (a) sandy, (b) fine
milky, and (c) ultrafine. The asterisks indicate extracellular matrix‐like layer on the
surface of the fine milky cells. (d) Switchgrass cell suspension cultures of the fine
milky type before digestion (10×) and (e) protoplasts isolated after digestion (20×).
(Reproduced with permission from Mazarei et al. (2011).)
Figure 5.14
Figure 5.14
Protoplasts
derived from
leaves of
Arabidopsis.
Summary
• Tissue culture is required for in vitro
manipulation of plant tissues and
transformation
• Auxins and cytokinins are the most
important hormones for tissue culture