Transcript AG-PSB-02.441-05.3p Tissue Culture

Plant Tissue Culture
Original by Linda Rist
Modified by Georgia Agricultural
Education Curriculum Office
July, 2002
August 2008
T.C.
 Refers to technique of
growing plant cells, tissues,
organs, seeds
or other plant parts in a
sterile environment on a
nutrient medium
August 2008
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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1930’s
White worked on T.C.
discovery of plant growth
regulators
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1930’s
importance of vitamins
was determined for shoot
and root culturing
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1930’s
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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60’s & 70’s
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
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4N
all chromosomes of both
plants
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70’s &80’s
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. don’t 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
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