Plant Tissue Culture
Denita Hadziabdic and Neal Stewart
Spring 2014
Outline

Introduction and history of tissue culture

Review of anatomy/morphology of plants

Tissue culture advantages and disadvantages

Stages of tissue culture

Media preparation

Plant growth regulators
Plant tissue culture definition
The growth and development of plant seeds,
organs, explants, tissues, cells or protoplasts
on nutrient media under sterile (axenic)
conditions.
Introduction to tissue culture
Alternative to conventional
propagation:



Establishment
Production
Maintenanced
-Production of plant organs and
tissues in aseptic culture
-Needed to enable the production of
Image courtesy of Wollemi Pine International
transgenic plants
History and 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 (TDNA) 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)
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
A
B
Style
Sepals
Ovary
Petals
5 carpels
Locules
Ovules
Ovary
Ovules
2 Carpels
Filaments
Filaments
Petals
Receptacle
Sepals
Cross section-Geranium sp.
C
Longitudinal section-Geranium sp.
D
Epidermis
Tepals
(sepals+
petals)
Stamens (6)
Endothecium
Ovary
Pollen
Vacscular
tissue
Cross section-Lilium sp.
Anther-Lilium sp.
Tapetum
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.
Tissue Culture Advantages and Uses
1. Mass propagation of specific clones
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
Why use micropropagation methods

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
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
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

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:
The physiological and morphological changes that
occur in a cell, tissue, or organ during
development.
Explant redefined
X-your-plant: excise a small piece of leaf, stem or other
tissue, and place them into tissue 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 nutrients and plant
growth regulators the explant
needs
Embryogenesis:
The formation of a zygote after fusion of a sperm and egg
cell. Have: seed coat, endosperm, and embryo
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 individual cells not from
pre-existing meristems.
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.
TC developmental stages
Stage 0 - Donor plant selection and preparation of
explants
Stage I - Establishment of aseptic cultures
Stage II - Proliferation of axillary shoots
Stage III - Pretransplant (rooting)
Stage IV - Transfer to natural environment
Stage 0 - Donor plant selection/preparation of 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
Stage I - Establishment of aseptic cultures

Indexing (screening) of donor plants

Establishment of sterile culture
 Surface


sterilization prior to placement in culture
Providing an in vitro environment that promotes
stable shoot production
 Addition
of cytokinins/auxins
pH needs
to be
~5.8
Stage II - Proliferation of axillary shoots

Repeated enhanced formation of axillary shoots
from shoot tips or lateral buds

Increased cytokinin concentrations (shoot
proliferation vs. shoot elongation)

Low or absent auxin levels

4-8 week subculturing intervals (1 cycle)
WPM 1 μM BA
MS 1 μM BA
WPM 2 μM BA
MS 2 μM BA
MS 0 μM BA
MS 2 μM BA
MS 1 μM BA
MS 4 μM BA
Stage III - Pretransplant (rooting)

Focused on rooting of shoots/shoot clusters from stage II

In vitro rooting for few weeks
  cytokinin  auxin

After rooting, transfer to ex vitro environment (stage IV)
 Roots not as sturdy as ex vitro roots
3 μM IBA
30 μM IBA
100 μM IBA
300 μM IBA
Percentage rooted (%)
Rooted microshoots
100
80
60
40
20
0
0μM IBA
3μM IBA
30μM IBA
100μM IBA
Rooted microshoots
300μM IBA
Stage IV - Transfer to natural environment

Acclimatization of microshoots from
heterotrophic (sucrose in jar) to
photoautotrophic (photosynthesis) stage

In vitro grown plants have:




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low photosynthetic activity
poor water loss control
abnormal stomata functioning
poor vascular connections between
roots and shoots
poorly differentiated mesophyll
What’s in tissue culture medium?

Water

Mineral salts

Carbon sources

Vitamins

Plant growth regulators

Other constituents as
desired
Plant growth regulators

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
TC Developmental Stages (Review)
Stage 0 - Donor plant selection and preparation
Stage I - Establishment of aseptic cultures
Stage II - Proliferation of axillary shoots
Stage III - Pretransplant (rooting)
Stage IV - Transfer to natural environment
Natural embryogenesis
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
Plant growth regulators (PGRs)

PGR’s are signal molecules, present in trace
quantities

Differ from animal hormones
Maintain a condition of
equilibrium/stability
 don't
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
Auxins: natural and synthetic
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
"The Power of Movement in
Plants“ (1880) by Charles
Darwin:
Image courtesy of North Carolina State University
Effects of light on movement
of canary grass Phalaris
canariensis coleoptiles
Experiment - the tip of the coleoptile perceived the light
- produced some signal
- signal was transported to the lower part of coleoptile
- induction of physiological response of bending
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
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)
Effect of different ratio of auxin to cytokinin
http://www.oup.com/uk/orc/bin/0199254680/ch02
Cytokinins: natural and synthetic
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
http://www.iesnz.co.nz/webfiles/IES/webpages/images/2185/ethylene
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
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