‘Green’ biotechnology

• Introduction
• DNA, Chromosomes, Genomes
• Plant Transformation
• Modern Plant Breeding
• Plant tissue culture
• Molecular Marker
 What is Plant Tissue Culture?

“… the aseptic culture of plant

protoplasts, cells, tissues or
organs under conditions
which lead to cell
multiplication or
regeneration of organs or
whole plants “
Totipotency ….
… each livinggenetic
complete cell has a complete
blue print genetic
blueprint and therefore has the potential to
develop into an entire plant.
……cells
cellslines
differentiate
differentiate to form specialised
tissues and organs

unlikeplant
… living animal cells,
cells canliving plant cells can
re-differentiate
de-differentiate and then re-differentiate to
form different cell types
Therefore,

tissue can be regenerated from

explants such as cotyledons,
hypocotyls, leaf, ovary, protoplast,
petiole, root, anthers, etc.
Tobacco plants from single cells

[Steeves & Sussex 1972]

Regeneration at
the microscopical
level
Organogenesis:
The process of initiation and
development of a structure that shows
natural organ form and/or function.

Embryogenesis:
The process of initiation and
development of embryos or embryo-like
structures from somatic cells (Somatic
embryogenesis).
 What for?

• Tissue culture produces clones, in

which all product cells have the same
genotype (unless affected by mutation
during culture)
 What for?

• A more recent advance is the use of

plant and animal tissue culture along
with genetic modification using viral and
bacterial vectors and gene guns to
create genetically engineered
organisms
Early Cell Culture ….

…Haberlandt .. early 1900’s

… proposed concept of totipotency
… cells cultured under right conditions

Callus cultured from tree cambium

(Gautheret, Nobecourt, Whire in the 1930s.

… cells kept alive but did not develop

 First commercial use
• The first commercial use of
plant clonal propagation on
artificial media was in the
germination and growth of
orchid plants, in the 1920’s

• In the 1950’s and 60’s there

was a great deal of research,
but it was only after the
development of a reliable
artificial medium (Murashige
& Skoog, 1962) that plant
Young cymbidium orchids
tissue culture really ‘took off’
commercially
 What is needed?
Tissue culture has several critical requirements:

• Appropriate tissue (some tissues culture better than

others)

• A suitable growth medium containing energy sources

and inorganic salts to supply cell growth needs. This can
be liquid or semisolid

• Aseptic (sterile) conditions, as microorganisms grow

much more quickly than plant and animal tissue and can
over run a culture
 What is Needed
• Growth regulators - in plants, both auxins
& cytokinins.

• Frequent subculturing to ensure adequate

nutrition and to avoid the build up of waste
metabolites
Culturing Plant Tissue - the steps

• Selection of the plant

tissue (explant) from a
healthy vigorous
‘mother plant’ - this is
often the apical bud,
but can be other tissue

• This tissue must be

sterilized to remove
microbial contaminants
Callus – an undifferentiated tissue
Elimination of microbial contaminants

Surface contaminants - principally microbial saprophytes

that are eliminated by surface disinfestation

Internal contaminants - principally pathogens that are

eliminated by thermotherapy (35-40 C) and culture of
explants free of organisms or by antibiotics

Maintenance of asepsis (free from microorganisms) - during

excision and culture - procedures are carried out in
sterile laminar flow positive pressure hoods (0.3 µm
filters)
Common Plant Tissue Disinfestants

Concentration of Time
Agent Active Ingredient Phytotoxicity (min)
Na hypochlorite
(Laundry Bleach) 0.25-1% Moderate 5-20
Ca hypochlorite 9-10% Moderate 5-20
H2O2 3-10% High 5-20
Alcohol
(ethanol or
isopropanol) 70% High <30 sec

These disinfestants can be used in combination and the

effectiveness of these solutions is enhanced by using a wetting agent
such as a detergent.
Culturing Plant Tissue - the steps
• Establishment of the
explant in a culture
medium. The medium
sustains the plant cells and
encourages cell division. It
can be solid or liquid

• Each plant species has

particular medium
requirements that must be
established by trial and
error
 Culture Medium constituents

• Inorganic salt formulations

• Source of carbohydrate
• Vitamins
• Water
• Plant hormones - auxins, cytokinins, GA’s
• Solidifying agents
Composition
of tissue
culture
medium is
complex
• Two Hormones Affect Plant
Differentiation:
– Auxin: Stimulates Root Development
– Cytokinin: Stimulates Shoot Development

• Generally, the ratio of these two

hormones can determine plant
development:
– ↑ Auxin ↓Cytokinin = Root Development
– ↑ Cytokinin ↓Auxin = Shoot Development
– Auxin = Cytokinin = Callus Development
2iP/IAA 0.5/2.5

Effect of
Kin/IAA 0.5/2.5 different
auxine and
Kin/IBA 0.5/2.5 cytokinine
concentration
Kin/IBA 0.5/0.5
on tissue
developemt

Kin/NAA 0.5/0.5
Culturing Plant Tissue - the steps

• The rooted shoots are

potted up (deflasked)
and ‘hardened off’ by
gradually decreasing
the humidity

• This is necessary as
many young tissue
culture plants have no
waxy cuticle to prevent
water loss
Factors Affecting Plant Tissue Culture
• Growth Media
– Minerals, Growth factors, Carbon source, Hormones
• Environmental Factors
– Light, Temperature, Photoperiod, Sterility, Media
• Explant Source
– Usually, the younger, less differentiated the explant,
the better for tissue culture
• Genetics
– Different species show differences in amenability to
tissue culture
– In many cases, different genotypes within a species
will have variable responses to tissue culture;
response to somatic embryogenesis has been
transferred between melon cultivars through sexual
hybridization
Why do Plant Tissue Culture?
• A single explant can be multiplied into several
thousand plants in less than a year - this
allows fast commercial propagation of new
cultivars

• Taking an explant does not usually destroy

the mother plant, so rare and endangered
plants can be cloned safely

• Once established, a plant tissue culture line

can give a continuous supply of young plants
throughout the year
 Why do Plant Tissue Culture? II
• In plants prone to virus diseases, virus free
explants (new meristem tissue is usually virus
free) can be cultivated to provide virus free
plants

• Plant ‘tissue banks’ can be frozen, then

regenerated through tissue culture

• Plant cultures in approved media are easier to

export than are soil-grown plants, as they are
pathogen free and take up little space (most
current plant export is now done in this
manner)
Tissue Culture Applications
- Micropropagation
- Germplasm preservation
- Somaclonal variation & mutation selection
- Embryo Culture
- Haploid & Dihaploid Production
- In vitro hybridization – Protoplast Fusion
- Industrial Products from Cell Cultures
Micropropagation
A single explant can be
multiplied into several
thousand plants in less than
a year - this allows fast
commercial propagation of
new cultivars
Features of Micropropagation
• Clonal reproduction
– Way of maintaining heterozygozity
• Multiplication stage can be recycled many
times to produce an unlimited number of clones
– Routinely used commercially for many ornamental
species, some vegetatively propagated crops
• Easy to manipulate production cycles
– Not limited by field seasons/environmental influences
• Disease-free plants can be produced
– Has been used to eliminate viruses from donor plants
Micropropagation

– generation of genetical
identical plants
 Micropropagation of almost all
the fruit crops and vegetables
is possible now

• Some examples: dwarfing sweet cherry,

Shade trees, Ornamental shrubs, Roses,
Clematis, Lilacs, Saskatoon berries,
Nutraceutical Plants, Rhododendron,
Azalea, mustard, corn, soybeans, wheat,
rice, cotton, tomato, potato, citrus, turf,
legumes
Micropropagation
- allows rapid
propagation of new varieties
- economical in time and space
- disease free
- elite propagules

example: violets
 Germplasm Preservation
Slow growth techniques

o e.g.: ↓ Temp., ↓ Light, media supplements

(osmotic inhibitors, growth retardants), tissue
dehydration, etc…
o Medium-term storage (1 to 4 years)
Germplasm Preservation

Example:
Titan Arum
(Amorphophallus titanum)
 Germplasm Preservation

Cryopreservation
o Ultra low temperatures (-196 °C)
o Stops cell division &
metabolic processes
o Very long-term (indefinite?)
Cryopreservation Requirements
• Storage
– Usually in liquid nitrogen (-196oC) to avoid changes in
ice crystals that occur above -100oC
• Thawing
– Usually rapid thawing to avoid damage from ice crystal
growth
• Recovery
- Thawed cells must be washed of cryoprotectants
and nursed back to normal growth
– Avoid callus production to maintain genetic stability
Cryopreservation Requirements
• Preculturing
– Usually a rapid growth rate to create cells with small
vacuoles and low water content
• Cryoprotection
– Glycerol, DMSO, PEG, etc…, to protect against ice
damage and alter the form of ice crystals
• Freezing
– The most critical phase; one of two methods:
• Slow freezing allows for cytoplasmic dehydration
• Quick freezing results in fast intercellular freezing with little
dehydration
 Embryo Culture
Embryo culture developed from the need
to rescue embryos (embryo rescue)
from wide crosses where fertilization
occurred, but embryo development did
not occur
 Embryo Culture Uses
• Rescue F1 hybrid from a wide cross
• Overcome seed dormancy, usually with
addition of hormone to media (GA)
• To overcome immaturity in seed
– To speed generations in a breeding program
– To rescue a cross or self (valuable genotype) from
dead or dying plant
 Embryo Culture Uses

Rescue F1 hybrid from a wide cross

Example: Anthurium
 Embryo Rescue Process
• Make cross between two species
• Dissect embryo (usually immature)
– The younger the embryo, the more difficult to culture
• Grow on culture medium using basic tissue culture
techniques, use for breeding if fertile
• Many times, resulting plants will be haploid because
of lack of pairing between the chromosomes of the
different species
– This can be overcome by doubling the chromosomes,
creating allotetraploids
 Embryo rescue process

15 days

30 days

50 days 80 days
Regeneration of grape plants via
somatic embryosgenesis
Potential uses for tissue culture
in plant breeding
• Eliminate virus from infected plant
selection

– Either via meristem culture or sometimes via

heat treatment of cultured tissue (or
combination)
 Phytosanitation

Bacteria or Virus
infected plant
Infection of shoot Regeneration of infected
meristem plants

In a number of plants the

shoot meristem doe‘s not Regeneration of healthy
get infected pathogen-free plants
Eliminate virus from infected plant selection.

often used for potato, strawberry, banana, citrus

Isolation of the shoot meristem

 Somaclonal Variation

• There are two general types of Somaclonal Variation:

– Heritable, genetic changes (alter the DNA)
– Stable, but non-heritable changes (alter gene
expression, epigenetic)

– used in mutation breeding

Somaclonal variability

Kohleria „Orange Glow“, eine durch mutagene

Behandlung von Gewebekulturen erhaltene Mutante
(Oben) im Vergleich zur Ausgangsform (links).
Somaclonal Breeding Procedures
• Use plant cultures as starting material
– Idea is to target single cells in multi-cellular culture
– Usually suspension culture, but callus culture can work
(want as much contact with selective agent as possible)
• Optional: apply physical or chemical mutagen
• Apply selection pressure to culture
– Target: very high kill rate, you want very few cells to
survive, so long as selection is effective
• Regenerate whole plants from surviving cells
Somaclonal/Mutation Breeding
• Advantages
– Screen very high populations (cell based)
– Can apply selection to single cells
• Disadvantages
– Many mutations are non-heritable
– Requires dominant mutation (or double recessive
mutation); most mutations are recessive
Industrial Products from Cell Cultures

• Secondary metabolites produced by plants

– Alkaloids, Terpenoids, Steroids, Anthocyanins,
Polyphenols
• Often unclear function in the plant
• Often restricted production (specific species,
tissue or organ)
• Many are commercially valuable
• Cell culture techniques allow large-scale
production of specific secondary metabolites
Secondary
metabolites
Example:
Production of Shikonin via cell
culture of Lithospermum Shikonin crystals
erythrorhizon

ムラサキ科 Shikonin-products
„bio-soap, bio-lipstick“
Tissue Culture Applications
- Micropropagation
- Germplasm preservation
- Somaclonal variation & mutation selection
- Embryo Culture
- Haploid & Dihaploid Production
- In vitro hybridization – Protoplast Fusion
- Industrial Products from Cell Cultures
Early tissue culture ….
- dependent on discovery of
“growth regulators”

] Cell enlargement … role of auxins

] Cell division ... role of cytokinins

] Regeneration from tobacco pith ..

(Skoog and Miller) … interaction of auxin
and cytokinin gives differentiation.
 Essential Nutrients

Macronutrients (required content in the plant - 0.1% or

% per dry weight) - C, H, O, P, K, N, S, Ca, Mg

Micronutrients (requirement - ppm/dry weight) - Fe,

Mn, Zn, Cu, B, Cl, Mo

Na, Se and Si are essential for some plants

 Hormone Balance
Auxin Cytokinin
High Low
Root formation on cuttings
Embryogenesis
Adventitious root formation in callus
Callus initiation
Adventitious shoot formation
Axillary shoot growth

Low High
 Further development …

] GA for growth of shoots

] Aux + Cyt + sucrose

> vascular development

] Culture of ‘thin layers’

… many interacting factors eg pH
 Carrot plants from root cells – Stewart in 1964

[Steeves & Sussex 1972]

Plant Organ Culture ….

Murashige and Skoog 1962 - mineral media

→ micropropagation
 2 Types of Cell & Tissues

DMany different types of cells

DVarying degrees of specialisation

- Meristematic
- Embryonic
- Reproductive
 Meristematic tissues ...

DShoot ... apical,

… axillary
 Culturing Plant Tissue - the steps

• Multiplication- The
explant gives rise to a
callus (a mass of
loosely arranged cells)
which is manipulated by
Dividing shoots varying sugar
concentrations and the
auxin (low): cytokinin
(high) ratios to form
multiple shoots
• The callus may be
subdivided a number of
times

Warmth and good light are essential

Culturing Plant Tissue - the steps

• Root formation - The

shoots are
transferred to a
growth medium with
relatively higher
auxin: cytokinin
ratios
The pottles on these racks
are young banana plants and
are
growing roots
 new leaf
tunica
apical
meristem
corpus
leaf trace

axillary
meristem
procambium

cortex pith
 Advantages Cont’d
• facilitates safer movements of germplasm
across nations - In vitro germplasm assures
the exchange of pest and disease free
material
• great for
– vegetatively reproduced crops
– crops which produce few seeds or highly
heterozygous seeds.
Vegetative Propagation in Nature
• Layering - a drooping lower branch contacts
the soil (pressed down by snow or
vegetation); roots form at point of soil
contact forming a new genetically identical
tree
• When trees of some species are cut down,
new shoots emerge from the stump
• strawberries spread through sending out
above-ground horizontal shoots called
runners, also called stolons.
 Successes of Somaclonal/Mutation Breeding
Herbicide Resistance and Tolerance
• Resistance: able to break-down or metabolize the herbicide –
introduce a new enzyme to metabolize the herbicide
• Tolerance: able to grow in the presence of the herbicide –
either ↑ the target enzyme or altered form of enzyme
– Most successful application of somaclonal breeding have been
herbicide tolerance
– Glyphosate resistant tomato, tobacco, soybean (GOX enzyme)
– Glyphosate tolerant petunia, carrot, tobacco and tomato (elevated EPSP
)
(enolpyruvyl shikimate phosphate synthase)

• But not as effective as altered EPSP enzyme (bacterial sources)

– Imazaquin (Sceptor) tolerant maize
• Theoretically possible for any enzyme-targeted herbicide – it’s
relatively easy to change a single enzyme by changing a single
gene
 Other Targets for Somaclonal Variation
• Specific amino acid accumulators
– Screen for specific amino acid production
– e.g. Lysine in cereals
• Abiotic stress tolerance
– Add or subject cultures to selection agent
– e.g.: salt tolerance, temperature stresses, etc…
• Disease resistance
– Add toxin or culture filtrate to growth media
– Examples shown on next slide →
 Disease Resistant Success using Somaclonal Variation

Crop Pathogen Toxin

Alfalfa Colletotrichum sp. Culture filtrate
Banana Fusarium sp. Fusaric acid
Coffee Colletotrichum sp. Partially purified culture filtrate
Maize Helminthosporium maydis T-toxin
Oat* Helminthosporium victoriae Victorin
Oilseed Rape* Phoma lingam Culture filtrate
Peach Xanthomonas sp. Culture filtrate
Potato** Phytophthora infestans Culture filtrate
Rice* Xanthomonas oryzae Culture filtrate
Sugarcane** Helminthosporium sp. Culture filtrate
Sugarcane** Helminthosporium sachari Partially purified HS toxin
Tobacco* Psedomonas tabaci Methionine-sulfoximine
Tobacco* Alternaria alternata Partially purified toxin
*Shown to be heritable through sexual propagation
**Shown to be stable through vegetative propagation
 Requirements for Somaclonal/Mutation Breeding
• Effective screening procedure
– Most mutations are deleterious
• With fruit fly, the ratio is ~800:1 deleterious to beneficial
– Most mutations are recessive
• Must screen M2 or later generations
• Consider using heterozygous plants?
– But some say you should use homozygous plants to be sure effect is
mutation and not natural variation
• Haploid plants seem a reasonable alternative if possible
– Very large populations are required to identify desired
mutation:
• Can you afford to identify marginal traits with replicates & statistics?
Estimate: ~10,000 plants for single gene mutant
• Clear Objective
– Can’t expect to just plant things out and see what happens;
relates to having an effective screen
– This may be why so many early experiments failed
Tissue Culture in Plant Breeding

• rescue crosses which would otherwise abort

• Only method of reproduction for sterile
plants such as triploids (e.g., bananas)
• combine desirable root characteristics with
desirable shoot characteristics (e.g., grapes)
• Haploid generation through anther/pollen
culture is an important area in crop
improvement