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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.

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development of superior cultivars
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
Tissue Culture Applications
  • Micropropagation
  • Germplasm preservation
  • Somaclonal variation
  • Haploid & dihaploid production
  • In vitro hybridization – protoplast fusion
features of 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
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
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
slide8

COMPARISON OF CONVENTIONAL & MICROPROPAGATION OF VIRUS INDEXED REGISTERED RED RASPBERRIES

  • 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 None
  • infection:
ways to eliminate viruses
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 viruses
Elimination of viruses

Plant from the field

Pre-growth in the greenhouse

Active

growth

Heat treatment

35oC / months

Adventitious

Shoot

formation

‘Virus-free’ Plants

Virus testing

Meristem culture

Micropropagation cycle

indirect somatic embryogenesis

Explant → Callus Embryogenic → Maturation → Germination

Indirect Somatic Embryogenesis

  • Callus induction
  • Embryogenic callus development
  • Maturation
  • Germination
induction
Induction
  • Auxins required for induction
    • Proembryogenic masses form
    • 2,4-D most used
    • NAA, dicamba also used
development
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
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
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
slide16

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
slide17

In vitro Collection

  • Use :
        • Recalcitrant seeds
        • Vegetatively propagated
        • Large seeds
  • Concern:
  • Security
  • Availability
  • cost
slide18

Ways to achieve slow growth

  • 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
slide19

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
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
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 requirements23
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
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)
slide26

Epigenetic

the 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
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
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
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 method
Bulbosum Method

Hordeum bulbosum

Wild relative

2n = 2X = 14

Hordeum vulgare

Barley

2n = 2X = 14

  • 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)

X

Embryo Rescue

Haploid Barley

2n = X = 7

H. Bulbosum chromosomes eliminated

bulbosum technique
Bulbosum technique
  • H. vulgare is the seed parent
  • zygote develops into an embryo with elimination of HB 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
Culture Medium
  • Mineral salts – K, Ca, N most important
  • Carbohydrate and osmotic pressure
  • Amino acids
  • Plant growth regulators
culture medium34
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 medium35
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
slide36

“Wide” crossing of wheat and rye requires embryo rescue and chemical treatment to double the number of chromosomes.

Triticale

haploid plant production
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
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
Somatic Hybridization

Development of hybrid plants through the fusion of somatic protoplasts of two different plant species/varieties

somatic hybridization technique
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

slide41

Isolation of Protoplast

(Separartion of protoplasts from plant tissue)

2. Enzymatic Method

1. Mechanical Method

mechanical method
Mechanical Method

Plant Tissue

CellsPlasmolysis

MicroscopeObservation of cells

Release of protoplasm

Cutting cell wall with knife

Collection of protoplasm

mechanical method43
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
Enzymatic Method

Leaf sterlization, removal of

epidermis

Plasmolysed

cells

Plasmolysed

cells

Pectinase +cellulase

Pectinase

Protoplasm

released

Release of

isolated cells

Protoplasm released

cellulase

Isolated

Protoplasm

enzymatic method45
Enzymatic Method
  • Used forvariety 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
slide46

Protoplast Fusion

(Fusion of protoplasts of two different genomes)

1. Spontaneous Fusion

2. Induced Fusion

Intraspecific

Intergeneric

Chemofusion

Mechanical

Fusion

Electrofusion

uses for protoplast fusion
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
Spontaneous Fusion
  • Protoplast fuse spontaneously during isolation process mainly due to physical contact
      • Intraspecific produce homokaryones
      • Intergeneric have no importance
induced fusion
Induced Fusion

Chemofusion- fusion induced by chemicals

  • Types of fusogens
    • PEG
    • NaNo3
    • Ca 2+ ions
    • Polyvinyl alcohol
induced fusion50
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
Possible Result of Fusion of Two Genetically Different Protoplasts

= chloroplast

= mitochondria

Fusion

= nucleus

heterokaryon

cybrid

hybrid

cybrid

hybrid

identifying desired fusions
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
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
Problem and Limitation of Somatic Hybridization
  • Application of protoplast technology requires efficient plant regeneration system.
  • The lack of an efficient selection method for fused product is sometimes a major problem.
  • The end-product after somatic hybridization is often unbalanced.
  • Development of chimaeric calluses in place of hybrids.
  • Somatic hybridization of two diploids leads to the formation of an amphiploids which is generally unfavorable.
  • Regeneration products after somatic hybridization are often variable.
  • It is never certain that a particular characteristic will be expressed.
  • Genetic stability.
  • Sexual reproduction of somatic hybrids.
  • Inter generic recombination.
true in vitro fertilization
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
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)
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