Plant Tissue Culture
Application
Development of
superior cultivars
 Germplasm storage
 Somaclonal variation
 Embryo rescue
 Ovule and ovary cultures
 Anther and pollen cultures
 Callus and protoplast culture
 Protoplasmic fusion
 In vitro screening
 Multiplication
Tissue Culture Applications
Micropropagation
Germplasm preservation
Somaclonal variation
Haploid & dihaploid production
In vitro hybridization – protoplast fusion
Micropropagation
Micropropagation advantages
 From one to many propagules rapidly
 Multiplication in controlled laboratorium conditions
 Continuous propagation year round
 Potential for disease-free propagules
 Inexpensive per plant once established
 Precise crop production scheduling
 Reduce stock plant space
Micropropagation disadvantages
 Specialized equipment/facilities required
 More technical expertise required
 Protocols not optimized for all species
 Plants produced may not fit industry standards
 Relatively expensive to set up
Micropropagation applications
 Rapid increase of stock of new varieties
 Elimination of diseases
 Cloning of plant types not easily propagated by
conventional methods (few offshoots/ sprouts/
seeds; date palms, ferns)
 Propagules have enhanced growth features
(multibranched character)
Methods of micropropagation
 Axillary branching
• >95% of all
micropropagation
• Genetically stable
• Simple and
straightforward
• Efficient but prone to
 Adventitious shoot
genetic instability
formation (organogenesis)
 Somatic embryogenesis
• Little used. Potentially
phenomenally efficient
Axillary shoot proliferation
Growth of axillary buds stimulated by cytokinin treatment; shoots
arise mostly from pre-existing meristems
 Clonal in vitro propagation by
repeated enhanced formation
of axillary shoots from shoottips or lateral meristems
cultured on media
supplemented with plant
growth regulators, usually
cytokinins.
 Shoots produced are either
rooted first in vitro or rooted
and acclimatized ex vitro
Steps of micropropagation
(axillary branching and adventitious shoot
formation)
• Stage 0 – Selection & preparation of the mother plant
Sterilization of the plant tissue
• Stage I - Initiation of culture
Explants placed into growth media
• Stage II - Multiplication
Explants transferred to shoot media; shoots can be constantly
divided
• Stage III - Rooting
Explants transferred to root media
• Stage IV - Transfer to soil
Explants returned to soil; hardened off
Clean Stock Program Used for
Commercial Potato
Procedures for cleaning virus infected clones and subsequent
generation of nuclear seed potatoes for distribution
Seed Potato Production
A
C
B
D
Shoots (A) from virus-free merstems multiplied in vitro (B) are transferred into soil
medium and grown in a screened greenhouse (C, D) to ward off insect vectors
Ways to eliminate viruses
 Heat treatment.
Plants grow faster than viruses at high temperatures.
 Meristemming.
Viruses are transported from cell to cell through
plasmodesmata and through the vascular tissue. Apical
meristem often free of viruses. Trade off between infection
and survival.
 Not all cells in the plant are infected.
Adventitious shoots formed from single cells can give virusfree shoots.
Elimination of viruses
Plant from the field
Pre-growth in the greenhouse
Active
growth
Heat treatment
35oC / months
‘Virus-free’ Plants
Meristem culture
Adventitious
Shoot
formation
Virus testing
Micropropagation cycle
Somatic Embryogenesis
Explant → Callus Embryogenic → Maturation → Germination
1.Callus induction
2. Embryogenic callus development
3.Maturation
4.Germination
Induction
• Auxins required for induction
– Proembryogenic masses form
– 2,4-D most used
– NAA, dicamba also used
Development
 Auxin must be removed for embryo development
 Continued use of auxin inhibits embryogenesis
 Stages are similar to those of zygotic embryogenesis
–
–
–
–
–
Globular
Heart
Torpedo
Cotyledonary
Germination (conversion)
Maturation
• Require complete maturation with apical
meristem, radicle, and cotyledons
• Often obtain repetitive embryony
• Storage protein production necessary
• Often require ABA for complete maturation
• ABA often required for normal embryo
morphology
– Fasciation
– Precocious germination
Germination
• May only obtain 3-5% germination
• Sucrose (10%), mannitol (4%) may be required
• Drying (desiccation)
– ABA levels decrease
– Woody plants
– Final moisture content 10-40%
• Chilling
– Decreases ABA levels
– Woody plants
Peanut somatic embryogenesis
Plant germplasm preservation
 In situ : Conservation in ‘normal’ habitat
–rain forests, gardens, farms
 Ex Situ :
–Field collection, botanical gardens
–Seed collections
–In vitro collection: Extension of micropropagation techniques
•Normal growth (short term storage)
•Slow growth (medium term storage)
•Cryopreservation (long term storage)
 DNA Banks
In vitro Collection
Potential advantages of in vitro methods:
 little space needs
 plants are free of pests, pathogens and
viruses (and will remain so)
 no transfer labor (under storage
conditions)
 stored cultures can be used as nuclear
stock for vegetative preservation
 international shipping restrictions are
lessened
1. no soil
2. pest-free plants
In vitro Collection
• Basic goals of an in vitro storage system
– to maintain genetic stability
– to keep in indefinite storage without loss of viability
– must be economical
• Two/three types of systems:
– Normal growth
– Slow growth
– Cryopreservation
Normal Growth
1. It can be done either on semi solid media or in
liquid media
2. It is similar to multiplication stage in micropropagation
3. It must be frequently sub-cultured
4. When axillary buds are used as explants, it is
considered as genetically stabile
Slow growth
It can store at least 1 semester and maximum 6 years
without sub-culturing
Ways to achieve slow growth:
 Addition of inhibitors or retardants
 Increasing osmotic potential of the media
 Manipulating storage temperature and light (cold
storage (1-9° C))
 Reducing light intensity
 Mineral oil overlay (callus)
 Reduced oxygen tension
Plant Growth Retardants
any chemicals that slow cell division and
elongation in shoot tissues
Cause plants to be shorter and more compact
 Interrupts cell division, stem elongation, and seed
head formation
Roots continue to grow
May reduce the natural Gibberellic acid
May produce more ethylene
Cold storage
storage at non-freezing temps, from 1-9° C
dependent on species.
storage of shoot cultures (stage I or II)
• works well for strawberries, potatoes, grapes, prob. many
more spp.
• transferred (to fresh medium) every 6 month or on a yearly
basis
Advantages:
 simple,
 high rates of survival,
 useful for micro-propagation (especially in periods of low
demand)
Disadvantages
• It may not be suitable for tropical, subtropical
species because of susceptibility of these to chill
injury
• It is an alternative with coffee – shoot cultures
transferred to a medium with reduced nutrients
and lacking sucrose
• It requires refrigeration, which is more
expensive than storage in cryopreservation
In vitro storage of 10 C
Cryopreservation
Storage of living tissues at ultra-low temperatures (-196°C)
Conservation of plant germplasm
• Vegetatively propagated species (root and tubers, ornamental, fruit trees)
• Recalcitrant seed species (Howea, coconut, coffee)
Conservation of tissue with specific characteristics
• Medicinal and alcohol producing cell lines
• Genetically transformed tissues
• Transformation/Mutagenesis competent tissues (ECSs)
Eradication of viruses (Banana, Plum)
Conservation of plant pathogens (fungi, nematodes)
Cryopreservation Steps
 Selection
 Excision of plant tissues or organs
 Culture of source material
 Select healthy cultures
 Apply cryo-protectants
 Pre-growth treatments
 Cooling/freezing
 Storage
 Warming & thawing
 Recovery growth
 Viability testing
 Post-thawing
Cryopreservation Requirements
• Preculturing
– Usually a rapid growth rate to create cells with small vacuoles
and low water content
• Cryoprotection
– Cryoprotectant (Glycerol, DMSO/dimetil sulfoksida, PEG)
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
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 cryo-protectants and nursed
back to normal growth
– Avoid callus production to maintain genetic stability
Somaclonal Variation
 Variation found in somatic cells dividing mitotically in culture
 A general phenomenon of all plant regeneration systems that
involve a callus phase
 Variation in trait(s) generated by use of a tissue-culture cycle
 Genetic variations in plants that have been produced by plant
tissue culture and can be detected as genetic or phenotypic traits
Two general types of Somaclonal Variation:
– Heritable, genetic changes (alter the DNA)
– Stable, but non-heritable changes (alter gene expression,
epigenetic)
Genetic (Heritable Variations)
• Pre-existing variations in the somatic cells of explant
• Caused by mutations and other DNA changes
• Occur at high frequency
Epigenetic
(Non-heritable Variations)
• Variations generated during tissue culture
• Caused by temporary phenotypic changes
• Occur at low frequency
Causes of Somaclonal
Variations
Biochemical
Cause
Physiological
Cause
Genetic Cause
Genetic Cause
1.
2.
3.
4.
5.
6.
Change in chromosome number
Change in chromosome structure
Gene Mutation
Extrachomosomal gene mutation
Transposable element activation
DNA sequence
DNA sequence
Change in DNA
 Detection of altered fragment size by using
Restriction enzyme
 Change in Protein
 Loss or gain in protein band
 Alteration in level of specific protein
 Methylation of DNA
 Methylation inactivates transcription
process
Advantages of Somaclonal
Variations
• Help in crop improvement
• Creation of additional genetic varitaions
• Increased and improved production of
secondary metabolites
• Selection of plants resistant to various toxins,
herbicides, high salt concentration and mineral
toxicity
• Suitable for breeding of perrenial species
Disadvantages of Somaclonal
Variations
• A serious disadvantage occurs in operations which require
clonal uniformity, as in the horticulture and forestry
industries where tissue culture is employed for rapid
propagation of elite genotypes
• Sometime leads to undesirable results
• Selected variants are random and genetically unstable
• Require extensive and extended field trials
• Not suitable for complex agronomic traits like yield and
quality
• May develop variants with pleiotropic effects which are
not true.
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)
– Generate screening dosage (lethal dosage is dependent upon
the expected number survive cells
• Regenerate whole plants from surviving cells
- Direct organogenesis or embryogenesis
Requirements for Somaclonal 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
Embryo Culture Uses
• Rescuing interspecific and intergeneric hybrids
– wide hybrids often suffer from early spontaneous abortion
– cause is embryo-endosperm failure
– Gossypium, Brassica, Linum, Lilium
• Production of monoploids
– useful for obtaining "haploids" of barley, wheat, other cereals
– the barley system uses Hordeum bulbosum as a pollen parent
Embryo Culture of Citrus
Coconut embryo culture
Bulbosum Method
Hordeum
vulgare
Barley
2n = 2X = 14
X
↓
Hordeum
bulbosum
Wild relative
2n = 2X = 14
Embryo Rescue
Haploid Barley
2n = X = 7
H. Bulbosum
chromosomes
eliminated
• This was once more efficient than microspore culture in creating
haploid barley
• Now, with an improved culture media (sucrose replaced by
maltose), microspore culture is much more efficient (~2000
plants per 100 anthers)
Bulbosum technique
Hordeum vulgare is the seed parent
zygote develops into an embryo with elimination of Hordeum
bulbosum chromosomes
eventually, only HV chromosomes are left
embryo is "rescued“ to avoid abortion
Excision of the immature embryo:




Hand pollination of freshly opened flowers
Surface sterilization – EtOH on enclosing structures
Dissection – dissecting under microscope necessary
Plating on solid medium – slanted media are often used to
avoid condensation
Culture Medium
– Mineral salts – K, Ca, N most important
– Carbohydrate and osmotic pressure
– Amino acids
– Plant growth regulators
Culture Medium
–Carbohydrate and osmotic pressure
»
»
»
»
2% sucrose works well for mature embryos
8-12% for immature embryos
transfer to progressively lower levels as embryo grows
alternative to high sucrose – auxin & cyt PGRs
– amino acids
» reduced N is often helpful
» up to 10 amino acids can be added to replace N salts, incl.
glutamine, alanine, arginine, aspartic acid, etc.
» requires filter-sterilizing a portion of the medium
Culture Medium
– natural plant extracts
»
»
»
coconut milk (liquid endosperm of coconut)
enhanced growth attributed to undefined hormonal factors
and/or organic compounds
others – extracts of dates, bananas, milk, tomato juice
– PGRs
»
»
»
»
globular embryos – require low conc. of auxin and cytokinin
heart-stage and later – usually none required
GA and ABA regulate "precocious germination“
GA promotes, ABA suppresses
“Wide” crossing of wheat and rye
requires embryo rescue and chemical
treatment to double the number of
chromosomes.
Triticale
Haploid Plant Production
 Embryo rescue of interspecific
crosses
– Creation of alloploids
 Anther culture/Microspore
culture
– Culturing of Anthers or
Pollen grains (microspores)
– Derive a mature plant from a
single microspore
 Ovule culture
– Culturing of unfertilized
ovules (macrospores)
Initiation from Stamens and Pistils
Stamen explant
Embryogenic callus
Callus formation from Callus formation from
connective tissue
filament tip
Embryo development
Embryo germination
Poliploidization
Specific Examples of DH uses
• Evaluate fixed progeny from an F1
– Can evaluate for recessive & quantitative traits
– Requires very large dihaploid population, since no prior selection
– May be effective if you can screen some qualitative traits early
• For creating permanent F2 family for molecular marker
development
• For fixing inbred lines (novel use?)
– Create a few dihaploid plants from a new inbred prior to going to
Foundation Seed (allows you to uncover unseen off-types)
• For eliminating inbreeding depression (theoretical)
– If you can select against deleterious genes in culture, and screen
very large populations, you may be able to eliminate or reduce
inbreeding depression
– e.g.: inbreeding depression has been reduced to manageable level
in maize through about 50+ years of breeding; this may reduce
that time to a few years for a crop like onion or alfalfa
Somatic Hybridization
Development of hybrid plants through the fusion of somatic
protoplasts of two different plant species/varieties
Somatic hybridization technique
1. isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties
3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regeneration of hybrid plants
Isolation of Protoplast
(Separartion of
1. Mechanical Method
protoplasts from plant tissue)
2. Enzymatic Method
Mechanical Method
Cells Plasmolysis
Plant Tissue
Microscope Observation of cells
Cutting cell wall with knife
Release of protoplasm
Collection of protoplasm
Mechanical Method
Used for vacuolated cells like onion bulb scale,
radish and beet root tissues
Low yield of protoplast
Laborious and tedious process
Low protoplast viability
Enzymatic Method
Leaf sterlization, removal of
epidermis
Plasmolysed
cells
Plasmolysed
cells
Pectinase +cellulase
Protoplasm released
Pectinase
Protoplasm
released
Release of
isolated cells
cellulase
Isolated
Protoplasm
Enzymatic Method
 Used for variety of tissues and organs including
leaves, petioles, fruits, roots, coleoptiles, hypocotyls,
stem, shoot apices, embryo microspores
 Mesophyll tissue - most suitable source
 High yield of protoplast
 Easy to perform
 More protoplast viability
Protoplast Fusion
(Fusion of protoplasts of two different genomes)
1. Spontaneous Fusion
Intraspecific
Intergeneric
2. Induced Fusion
Chemofusion
Mechanical
Fusion
Electrofusion
Uses for Protoplast Fusion
 Combine two complete genomes
– Another way to create allopolyploids
 In vitro fertilization
 Partial genome transfer
– Exchange single or few traits between species
– May or may not require ionizing radiation
 Genetic engineering
– Micro-injection, electroporation, Agrobacterium
 Transfer of organelles
– Unique to protoplast fusion
– The transfer of mitochondria and/or chloroplasts between
species
Spontaneous Fusion
• Protoplast fuse spontaneously during isolation
process mainly due to physical contact
• Intraspecific produce homokaryones
• Intergeneric have no importance
Induced Fusion
Chemofusion- fusion induced by chemicals
• Types of fusogens
•
•
•
•
PEG
NaNo3
Ca 2+ ions
Polyvinyl alcohol
Induced Fusion
• Mechanical Fusion- Physical fusion of protoplasts
under microscope by using micromanipulator and
perfusion micropipette
• Electrofusion- Fusion induced by electrical stimulation
• Fusion of protoplasts is induced by the application of high strength
electric field (100kv m-1) for few microsecond
Possible Result of Fusion of Two
Genetically Different Protoplasts
= chloroplast
= mitochondria
Fusion
= nucleus
heterokaryon
cybrid
hybrid
hybrid
cybrid
Identifying Desired Fusions
• Complementation selection
– Can be done if each parent has a different selectable marker (e.g.
antibiotic or herbicide resistance), then the fusion product
should have both markers
• Fluorescence-activated cell sorters
– First label cells with different fluorescent markers; fusion
product should have both markers
• Mechanical isolation
– Tedious, but often works when you start with different cell types
• Mass culture
– Basically, no selection; just regenerate everything and then screen
for desired traits
Advantages of somatic
hybridization
• Production of novel interspecific and intergenic hybrid
– Pomato (Hybrid of potato and tomato)
• Production of fertile diploids and polypoids from sexually
sterile haploids, triploids and aneuploids
• Transfer gene for disease resistance, abiotic stress
resistance, herbicide resistance and many other quality
characters
• Production of heterozygous lines in the single species
which cannot be propagated by vegetative means
• Studies on the fate of plasma genes
• Production of unique hybrids of nucleus and cytoplasm
Problem and Limitation of
Somatic Hybridization
1. Application of protoplast technology requires efficient plant
regeneration system.
2. The lack of an efficient selection method for fused product is
sometimes a major problem.
3. The end-product after somatic hybridization is often unbalanced.
4. Development of chimaeric calluses in place of hybrids.
5. Somatic hybridization of two diploids leads to the formation of an
amphiploids which is generally unfavorable.
6. Regeneration products after somatic hybridization are often variable.
7. It is never certain that a particular characteristic will be expressed.
8. Genetic stability.
9. Sexual reproduction of somatic hybrids.
10. Inter generic recombination.
TYPICAL SUSPENSION PROTOPLAST
+ LEAF PROTOPLAST PEG-INDUCED
FUSION
NEW SOMATIC HYBRID PLANT
True in vitro fertilization
A procedure that involves retrieval of eggs and
sperm from the male and female and placing them
together in a laboratory dish to facilitate
fertilization
 Using single egg and sperm cells and fusing them
electrically
 Fusion products were cultured individually in 'Millicell'
inserts in a layer of feeder cells
 The resulting embryo was cultured to produce a fertile
plant
Requirements for plant genetic
transformation
• Trait that is encoded by a single gene
• A means of driving expression of the gene in
plant cells (Promoters and terminators)
• Means of putting the gene into a cell (Vector)
• A means of selecting for transformants
• Means of getting a whole plant back from the
single transformed cell (Regeneration)