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

1930s
White worked on T.C.
discovery of plant growth
regulators

1930s
importance of vitamins was
determined for shoot and root
culturing

1930s
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

60s & 70s

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

70s &80s
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. dont 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