plant tissue culture for pdf - جامعة نزوى...factors affecting plant tissue culture •...
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‘Green’ biotechnology• Introduction• DNA, Chromosomes, Genomes• Plant Transformation• Modern Plant Breeding• Plant tissue culture • Molecular Marker
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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 “
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Totipotency ….… each living cell has a complete genetic blueprint and therefore has the potential to develop into an entire plant.
… cells lines differentiate to form specialisedtissues and organs
… unlike animal cells, living plant cells can de-differentiate and then re-differentiate to form different cell types
… complete genetic blue print
… cells differentiate
… living plant cells can re-differentiate
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tissue can be regenerated from explants such as cotyledons,
hypocotyls, leaf, ovary, protoplast, petiole, root, anthers, etc.
Therefore,
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Tobacco plants from single cells
[Steeves & Sussex 1972]
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Regeneration at the microscopical level
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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).
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• Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture)
What for?
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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
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…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
Early Cell Culture ….
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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 tissue culture really ‘took off’commercially
Young cymbidium orchids
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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
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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
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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
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Callus – an undifferentiated tissue
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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)
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Concentration of Time Agent Active Ingredient Phytotoxicity (min)Na hypochlorite(Laundry Bleach) 0.25-1% Moderate 5-20Ca hypochlorite 9-10% Moderate 5-20H2O2 3-10% High 5-20Alcohol(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.
Common Plant Tissue Disinfestants
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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
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Culture Medium constituents
• Inorganic salt formulations• Source of carbohydrate• Vitamins• Water• Plant hormones - auxins, cytokinins, GA’s• Solidifying agents
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Composition of tissue culture medium is complex
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• 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
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Effect of different auxine andcytokinineconcentration on tissue developemt
2iP/IAA 0.5/2.5
Kin/IAA 0.5/2.5
Kin/IBA 0.5/2.5
Kin/IBA 0.5/0.5
Kin/NAA 0.5/0.5
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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
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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
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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
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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)
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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
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MicropropagationA single explant can be multiplied into several thousand plants in less than a year - this allows fast commercial propagation of new cultivars
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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
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Micropropagation
– generation of geneticalidentical plants
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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
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Micropropagation- allows rapidpropagation of new varieties
- economical in time and space - disease free - elite propagules
example: violets
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Germplasm PreservationSlow growth techniques
o e.g.: ↓ Temp., ↓ Light, media supplements (osmotic inhibitors, growth retardants), tissue dehydration, etc…
o Medium-term storage (1 to 4 years)
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Germplasm Preservation
Example:Titan Arum (Amorphophallus titanum)
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Germplasm Preservation
Cryopreservationo Ultra low temperatures (-196 °C)o Stops cell division &
metabolic processeso Very long-term (indefinite?)
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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 cryoprotectantsand nursed back to normal growth– Avoid callus production to maintain genetic stability
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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
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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
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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
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Embryo Culture Uses
Example: Anthurium
Rescue F1 hybrid from a wide cross
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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
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Embryo rescue process
15 days
30 days
50 days 80 days
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Regeneration of grape plants via somatic embryosgenesis
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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)
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Phytosanitation
Bacteria or Virus infected plant
Infection of shoot meristem
In a number of plants the shoot meristem doe‘s not get infected
Regeneration of infected plants
Regeneration of healthy pathogen-free plants
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Isolation of the shoot meristem
Eliminate virus from infected plant selection.
often used for potato, strawberry, banana, citrus
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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
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Somaclonal variability
Kohleria „Orange Glow“, eine durch mutagene Behandlung von Gewebekulturen erhaltene Mutante (Oben) im Vergleich zur Ausgangsform (links).
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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
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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
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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
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Secondary metabolitesExample:Production of Shikonin via cell culture of Lithospermumerythrorhizon
Shikonin crystals
Shikonin-products„bio-soap, bio-lipstick“
ムラサキ科
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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
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] Cell enlargement … role of auxins] Cell division ... role of cytokinins
- dependent on discovery of“growth regulators”
Early tissue culture ….
] Regeneration from tobacco pith ..
(Skoog and Miller) … interaction of auxin
and cytokinin gives differentiation.
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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
Essential Nutrients
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Hormone BalanceAuxin CytokininHigh Low
Low High
Root formation on cuttings Embryogenesis
Adventitious root formation in callus Callus initiation
Adventitious shoot formation Axillary shoot growth
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] GA for growth of shoots
] Aux + Cyt + sucrose> vascular development
] Culture of ‘thin layers’… many interacting factors eg pH
Further development …
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Carrot plants from root cells – Stewart in 1964
[Steeves & Sussex 1972]
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Plant Organ Culture ….
Murashige and Skoog 1962 - mineral media
→ micropropagation
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Many different types of cells
Varying degrees of specialisation
- Meristematic- Embryonic- Reproductive
2 Types of Cell & Tissues
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Shoot ... apical, … axillary
Meristematic tissues ...
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Culturing Plant Tissue - the steps
• Multiplication- The explant gives rise to a callus (a mass of loosely arranged cells) which is manipulated by varying sugar concentrations and the auxin (low): cytokinin(high) ratios to form multiple shoots
• The callus may be subdivided a number of times
Dividing shoots
Warmth and good light are essential
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Culturing Plant Tissue - the steps
• Root formation - The shoots are transferred to a growth medium with relatively higherauxin: cytokinin ratios
The pottles on these racks are young banana plants and aregrowing roots
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leaf trace
procambium
tunicanew leaf
corpus
pithcortex
apical meristem
axillarymeristem
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Advantages Cont’d
• facilitates safer movements of germplasmacross 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.
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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.
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Successes of Somaclonal/Mutation BreedingHerbicide 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
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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 →
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Disease Resistant Success using Somaclonal Variation
Partially purified toxinAlternaria alternataTobacco*
*Shown to be heritable through sexual propagation**Shown to be stable through vegetative propagation
Methionine-sulfoximinePsedomonas tabaciTobacco*
Partially purified HS toxinHelminthosporium sachariSugarcane**
Culture filtrateHelminthosporium sp.Sugarcane**
Culture filtrateXanthomonas oryzaeRice*
Culture filtratePhytophthora infestansPotato**
Culture filtrateXanthomonas sp.Peach
Culture filtratePhoma lingamOilseed Rape*
VictorinHelminthosporium victoriaeOat*
T-toxinHelminthosporium maydisMaize
Partially purified culture filtrateColletotrichum sp.Coffee
Fusaric acidFusarium sp.Banana
Culture filtrateColletotrichum sp.Alfalfa
ToxinPathogenCrop
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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
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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