plant tissue culture application
Post on 10-Feb-2016
80 Views
Preview:
DESCRIPTION
TRANSCRIPT
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 ApplicationsMicropropagation
Germplasm preservationSomaclonal variation
Haploid & dihaploid productionIn vitro hybridization – protoplast
fusion
Micropropagation
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
Microcutting propagation• It involves the production of shoots from
pre-existing meristems only.• Requires breaking apical dominance
• This is a specialized form of organogenesis
Steps of Micropropagation• Stage 0 – Selection & preparation of the mother
plant– sterilization of the plant tissue takes place
• Stage I - Initiation of culture– explant placed into growth media
• Stage II - Multiplication– explant transferred to shoot media; shoots
can be constantly divided• Stage III - Rooting
– explant transferred to root media• Stage IV - Transfer to soil
– explant returned to soil; hardened off
Conventional Micropropagation
Duration: 6 years 2 years
Labor: Dig & replant every 2 years; Subculture every 4 weeks;
unskilled (Inexpensive) skilled (more expensive)
Space: More, but less expensive (field) Less, but more expensive (laboratory)
Required to prevent viral Screening, fumigation, spraying Noneinfection:
COMPARISON OF CONVENTIONAL & MICROPROPAGATION OF VIRUS
INDEXED REGISTERED RED RASPBERRIES
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 virus-free shoots.
Elimination of virusesPlant from the field
Pre-growth in the greenhouse
‘Virus-free’ Plants
Heat treatment35oC / months
Activegrowth
Meristem culture
Micropropagation cycle
Virus testing
AdventitiousShoot formation
Indirect Somatic EmbryogenesisExplant → Callus Embryogenic → Maturation →
Germination
1.Callus induction2. Embryogenic callus
development3.Maturation
4.Germination
Induction• Auxins required for induction
–Proembryogenic masses form–2,4-D most used–NAA, dicamba also used
DevelopmentAuxin must be removed for embryo
developmentContinued use of auxin inhibits embryogenesisStages 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
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
Plant germplasm preservation
Use : Recalcitrant seeds Vegetatively
propagated Large seeds
In vitro Collection
Concern: SecurityAvailabilitycost
Use of immature zygotic embryos (not for vegetatively propagated species)
Addition of inhibitors or retardants Manipulating storage temperature and
light Mineral oil overlay
Reduced oxygen tension Defoliation of shoots
Ways to achieve slow growth
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)
CryopreservationStorage of living tissues at ultra-low temperatures
(-196°C)
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
Some mechanisms: Karyotipic alteration Sequence variation
Variation in DNA Methylation
Two general types of Somaclonal Variation:– Heritable, genetic changes (alter the DNA)– Stable, but non-heritable changes (alter gene
expression, epigenetic)
Epigeneticthe study of gene regulation that does not involve making changes to the SEQUENCE of the DNA,
but rather to the actual BASES within the nucleotides and to the HISTONES
The three main mechanisms for regulation are: CpG island methylation (…meCGmeCGmeCGmeCGmeCGmeCGmeCGmeCG…) acetylation and methylation of histone H3 the production of antisense RNA
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
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
– 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
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
Bulbosum MethodHordeum vulgareBarley
2n = 2X = 14
Hordeum bulbosum
Wild relative2n = 2X = 14
Haploid Barley2n = X = 7
H. Bulbosum chromosomes
eliminated
X
Embryo Rescue↓
• 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 techniqueH. vulgare is the seed parentzygote develops into an embryo with elimination
of HB chromosomeseventually, only HV chromosomes are leftembryo 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
– 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
Culture Medium
“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)
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 HybridizationDevelopment of hybrid plants through the
fusion of somatic protoplasts of two different plant species/varieties
Somatic hybridization technique
1. isolation of protoplast1. isolation of protoplast
2. Fusion of the protoplasts of desired species/varieties2. Fusion of the protoplasts of desired species/varieties
3. Identification and Selection of somatic hybrid cells3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells4. Culture of the hybrid cells
5. Regeneration of hybrid plants 5. Regeneration of hybrid plants
Isolation of Protoplast (Separartion of protoplasts from plant tissue))
1. Mechanical Method 2. Enzymatic Method
Mechanical Method
Plant Tissue
Collection of protoplasm
Cells Plasmolysis
Microscope Observation of cells
Cutting cell wall with knife Release of protoplasm
Mechanical Method
Used for vacuolated cells like onion bulb scale, radish and beet root tissues
Low yield of protoplastLaborious and tedious processLow protoplast viability
Enzymatic Method
Leaf sterlization, removal of epidermis
Plasmolysed cells
Plasmolysed cells
Pectinase +cellulase Pectinase
Protoplasm released Release of isolated cells
cellulase
Protoplasm released
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 FusionProtoplast Fusion(Fusion of protoplasts of two different genomes(Fusion of protoplasts of two different genomes))
1. Spontaneous Fusion 2. Induced Fusion
Intraspecific Intergeneric ElectrofusionMechanical Fusion
Chemofusion
Uses for Protoplast FusionCombine two complete genomes
– Another way to create allopolyploids In vitro fertilizationPartial 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
• Types of fusogens• PEG• NaNo3
• Ca 2+ ions• Polyvinyl alcohol
Chemofusion- fusion induced by chemicals
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
= nucleusFusion
heterokaryon
cybrid cybridhybrid hybrid
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
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
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
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)
top related