Plant Tissue Culture

T.C.
☛ Refers to technique of growing
plant cells, tissues, organs, seeds
☛or other plant parts in a sterile
environment on a nutrient
medium
 History
☛In 1902 Haberlandt
proposed that single plant
cells could be cultured
 Haberlandt
☛did not culture them himself
 1930’s
☛White worked on T.C.
☛discovery of plant growth
regulators
 1930’s
☛importance of vitamins was
determined for shoot and root
culturing
 1930’s
☛Indole-Acetic Acid
☛IAA
☛discovered in 1937
 IAA
☛2,4-D
☛Dicamba
☛NAA
☛IBA
☛all synthetic hormones
 1957-58
☛Miller and Skoog
☛University of Wisconsin -
Madison
☛discovered Kinetin
 Kinetin
☛a cytokinin
☛plays active role in
organogenesis
 1958
☛Steward developed somatic
embryo from carrot cells
 1958-60
☛Morel cultured orchids and
dahlias
☛freed them from a viral
disease
 1962
☛Murashige and Skoog
☛published recipe for MS
Medium
 60’s & 70’s
☛Murashige cloned plants in
vitro
☛promoted development of
commercial plant T.C. labs
 1966
☛raised haploid plants from
pollen grains
 1972
☛used protoplast fusion to
hybridize 2 species of
tobacco into one plant
☛contained 4N
 4N
☛all chromosomes of both
plants
 70’s &80’s
☛develop techniques to
introduce foreign DNA into
plant cells
☛beginning of genetic
engineering
 T.C. Media
☛functions
☛provide H2O
☛provide mineral nutritional
needs
 T.C. Media
☛provide growth regulators
☛Provide vitamins
☛provide organic compounds
 T.C. Media
☛provide access to
atmosphere for gas exchange
☛serve as a dumping ground
for plant metabolites
 T.C. Media
☛H2O is usually distilled
☛minerals must provide 17
essential elements
☛energy source and carbon
skeletons - sucrose is preferred
 Vitamins
☛thiamine
☛pyridoxin
☛nicotinic acid
☛biotin
 Vitamins
☛citric acid
☛ascorbic acid
☛inositol
 Growth Regulators
☛auxins and cytokinins
☛gibberellic acid
☛abscissic acid
 pH of media
☛usually 5.0-5.7
 Media
☛must be sterile
☛autoclave at 250 F at 15 psi
for 15 minutes
 T.C. Stages
☛Explanting- Stage I
☛get plant material in sterile
culture so it survives
☛provide with nutritional and
light needs for growth
 Stage II
☛rapid multiplication
☛stabilized culture
☛goal for a commercial lab
☛difficult and time consuming
to maintain
 Stage II
☛occurs in different pathways
in different plants
 Rooting - Stage III
☛may occur in Stage II
☛usually induced by changes in
hormonal environment
☛lower cytokinin concentration and
increase auxin
 Rooting
☛may skip stage III and root
in a greenhouse
 Stage IV
☛transplantation and aftercare
☛usually done in greenhouse
☛keep RH high (relative
humidity)
 Stage IV
☛gradually increase light
intensity and lower RH after
rooting occurs
☛allows plants to harden and
helps plants form cuticle
 Cuticle
☛waxy substance promotes
development of stomates
☛plants in T.C. don’t have
cuticle
 Explant
☛portion of plant removed and used
for T.C.
☛Important features
☛size
☛source - some tissues are better
than others
 Explant
☛species dependent
☛physiological age - young
portions of plant are most
successful
 Explant
☛degree of contamination
☛external infestation - soak
plant in sodium hypochlorite
solution
 Explant
☛internal infection - isolate
cell that is not infected
☛roots - especially difficult
because of soil contact
 Explant
☛herbaceous plants
☛soft stem
☛easier to culture than woody
plants
 Patterns of
multiplication
☛stage II - light 100-300 foot
candles
☛callus - shoots - roots
☛stage III - rooting - light intensity
1000-3000 foot candles
 Genetic
transformation
☛permanent incorporation of
new or foreigh DNA into
genome of cell
 Transformation
methods
☛protoplast fusion
☛cell wall is enzymatically
removed from cell
 Protoplasts
☛naked plant cells
☛from 2 different plants can
be mixed together and forced
to fuse
 Protoplast fusion
☛results in heterokaryon
☛cell containing two or more
nuclei from different cells
☛homokaryon - from same
cell
 Protoplast fusion
☛allowed to regenerate cell
wall and then grow into
callus
☛callus turns to shoots
 Shotgun approach
☛DNA coated micro bullets
of gold or tungston
☛shot into growing cells
☛DuPont holds the patent
 Shotgun approach
☛injures cells
☛random success rate
 PEG
☛Polyethylene glycol
☛pores open similar to
electroporation
 Ti Plasmids
☛Tumor inducing
☛Agrobacterium temefasciens
☛infect cells with
agrobacterium which
contains desired DNA
 Ti Plasmids
☛monocots resist
agrobacterium infection
☛researchers are working to
overcome this
 Luciferase
☛an enzyme
☛put into tobacco using Ti
plasmid
 Luciferase
☛when transformed tobacco plants
are watered with solution
containing Luciferin
☛they break it down and emit light
 Luciferase
☛glowing in the dark
☛like a fire fly
Screening techniques
☛used to identify if culture
has taken on desired new
trait
 Examples
☛sensitivity to antibiotics
☛color
☛sensitivity to excess
deficiencies of substances in
growth media
 Conventional
☛plant breeding
☛egg cell gives half the
chromosomes and almost all of the
cytoplasm
☛male only gives its chromosomes
 Cont…….
☛This condition is called
maternal cytoplasmic
inheritance
 Microinjection
☛single cells from culture are
held stationary with gentle
suction
☛injected with a tiny syringe
loaded with DNA
 Microinjection
☛done under electron
microscope
 Electroporation
☛desired DNA in solution
outside cell
☛high energy pulses - 50,000
volts
☛for a millisecond
 Electroporation
☛cause tiny pores to open
☛allows DNA to enter the cell