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Plant Tissue Culture Application. Development of superior cultivars. Germplasm storage Embryo rescue Ovule and ovary cultures Anther and pollen cultures Callus and protoplast culture Protoplasmic fusion Plant Genetic Engineering. Tissue Culture Applications. Micropropagation

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development of superior cultivars
Development of superior cultivars
  • Germplasm storage
  • Embryo rescue
  • Ovule and ovary cultures
  • Anther and pollen cultures
  • Callus and protoplast culture
  • Protoplasmic fusion
  • Plant Genetic Engineering
tissue culture applications
Tissue Culture Applications
  • Micropropagation
  • Germplasm preservation
  • Somaclonal variation
  • Haploid & dihaploid production
  • In vitro hybridization – protoplast fusion
  • Plant genetic engineering
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


  • 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



Heat treatment

35oC / months




‘Virus-free’ Plants

Virus testing

Meristem culture

Micropropagation cycle


Storage of Plant germplasm

  • 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

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

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


  • 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 cryoprotectants
  • Pregrowth 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, 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 requirements1
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 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
  • 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
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


Isolation of Protoplast

(Separartion of protoplasts from plant tissue)

2. Enzymatic Method

1. Mechanical Method

mechanical method
Mechanical Method

Plant Tissue


MicroscopeObservation of cells

Release of protoplasm

Cutting cell wall with knife

Collection of protoplasm

mechanical method1
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






Pectinase +cellulase




Release of

isolated cells

Protoplasm released




enzymatic method1
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

Protoplast Fusion

(Fusion of protoplasts of two different genomes)

1. Spontaneous Fusion

2. Induced Fusion







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 fusion1
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


= nucleus






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.
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)