Plant Tissue Culture

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Plant Tissue Culture

• Matt Jakubik

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T.C.

• Refers to technique of growing plant cells,
  tissues, organs, seeds
• or other plant parts in a sterile environment on
  a nutrient medium

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History

• In 1902 Haberlandt proposed that single plant
  cells could be cultured

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Haberlandt

• did not culture them himself

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1930s

• White worked on T.C.
• discovery of plant growth regulators

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1930s

• importance of vitamins was determined for shoot
  and root culturing
• A, D, E, K, C, and B Complex

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1930s

• Indole-Acetic Acid
• IAA
• discovered in 1937

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IAA

• 2,4-D
• Dicamba
• NAA
• IBA
• all synthetic hormones

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1957-58

• Miller and Skoog
• University of Wisconsin - Madison
• discovered Kinetin

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Kinetin

• a cytokinin
• plays active role in organogenesis

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1958

• Steward developed somatic embryo from carrot cells

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1958-60

• Morel cultured orchids and dahlias
• freed them from a viral disease

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1962

• Murashige and Skoog
• published recipe for MS Medium

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60s 70s

• Murashige cloned plants in vitro
• promoted development of commercial plant T.C. labs

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1966

• raised haploid plants from pollen grains

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1972

• used protoplast fusion to hybridize 2 species of
  tobacco into one plant
• contained 4N
• all chromosomes of both plants

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70s 80s

• develop techniques to introduce foreign DNA into
  plant cells
• beginning of genetic engineering

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T.C. Media

• functions
• provide H2O
• provide mineral nutritional needs

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T.C. Media

• provide growth regulators
• Provide vitamins
• provide organic compounds

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T.C. Media

• provide access to atmosphere for gas exchange
• serve as a dumping ground for plant metabolites

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T.C. Media

• H2O is usually distilled
• minerals must provide 17 essential elements
• energy source and carbon skeletons - sucrose is
  preferred

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Vitamins

• thiamine
• pyridoxin
• nicotinic acid
• biotin

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Vitamins

• citric acid
• ascorbic acid
• inositol

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Growth Regulators

• auxins and cytokinins
• gibberellic acid
• abscissic acid

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pH of media

• usually 5.0-5.7

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Media

• must be sterile
• autoclave at 250 F at 15 psi for 15 minutes

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T.C. Stages

• Explanting- Stage I
• get plant material in sterile culture so it
  survives
• provide with nutritional and light needs for
  growth

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Stage II

• rapid multiplication
• stabilized culture
• goal for a commercial lab
• difficult and time consuming to maintain

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Stage II

• occurs in different pathways in different plants

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Rooting - Stage III

• may occur in Stage II
• usually induced by changes in hormonal
  environment
• lower cytokinin concentration and increase auxin

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Rooting

• may skip stage III and root in a greenhouse

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Stage IV

• transplantation and aftercare
• usually done in greenhouse
• keep RH high (relative humidity)

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Stage IV

• gradually increase light intensity and lower RH
  after rooting occurs
• allows plants to harden and helps plants form
  cuticle

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Cuticle

• waxy substance promotes development of stomates
• plants in T.C. dont have cuticle

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Explant

• portion of plant removed and used for T.C.
• Important features
• size
• source - some tissues are better than others

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Explant

• species dependent
• physiological age - young portions of plant are
  most successful

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Explant

• degree of contamination
• external infestation - soak plant in sodium
  hypochlorite solution

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Explant

• internal infection - isolate cell that is not
  infected
• roots - especially difficult because of soil
  contact

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Explant

• herbaceous plants
• soft stem
• easier to culture than woody plants

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Patterns of multiplication

• stage II - light 100-300 foot candles
• callus - shoots - roots
• stage III - rooting - light intensity 1000-3000
  foot candles

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Genetic transformation

• permanent incorporation of new or foreign DNA
  into genome of cell

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Transformation methods

• protoplast fusion
• cell wall is removed by enzymes from cell

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Protoplasts

• naked plant cells
• from 2 different plants can be mixed together and
  forced to fuse

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Protoplast fusion

• results in heterokaryon
• cell containing two or more nuclei from different
  cells
• homokaryon - from same cell

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Protoplast fusion

• allowed to regenerate cell wall and then grow
  into callus
• callus turns to shoots

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Shotgun approach

• DNA coated micro bullets of gold or tungston
• shot into growing cells
• DuPont holds the patent

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Shotgun approach

• injures cells
• random success rate

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PEG

• Polyethylene glycol
• pores open similar to electroporation

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Ti Plasmids

• Tumor inducing
• Agrobacterium temefasciens
• infect cells with agrobacterium which contains
  desired DNA

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Ti Plasmids

• monocots resist agrobacterium infection
• researchers are working to overcome this

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Luciferase

• an enzyme
• put into tobacco using Ti plasmid

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Luciferase

• when transformed tobacco plants are watered with
  solution containing Luciferin
• they break it down and emit light

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Luciferase

• glowing in the dark
• like a fire fly

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Screening techniques

• used to identify if culture has taken on desired
  new trait

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Examples

• sensitivity to antibiotics
• color
• sensitivity to excess deficiencies of substances
  in growth media

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Conventional

• plant breeding
• egg cell gives half the chromosomes and almost
  all of the cytoplasm
• male only gives its chromosomes

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Cont.

• This condition is called maternal cytoplasmic
  inheritance

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Microinjection

• single cells from culture are held stationary
  with gentle suction
• injected with a tiny syringe loaded with DNA

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Microinjection

• done under electron microscope

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Electroporation

• desired DNA in solution outside cell
• high energy pulses - 50,000 volts
• for a millisecond

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Electroporation

• cause tiny pores to open
• allows DNA to enter the cell