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Tissue culture
1.
Plant Tissue Culture
2.
T.C. Refers to
technique of growing plant cells, tissues, organs, seeds or other plant parts in a sterile environment on a nutrient medium
3.
History In 1902 Haberlandt
proposed that single plant cells could be cultured
4.
Haberlandt did not culture
them himself
5.
1930’s White worked on
T.C. discovery of plant growth regulators
6.
1930’s importance of vitamins
was determined for shoot and root culturing
7.
1930’s Indole-Acetic Acid IAA discovered in
1937
8.
IAA 2,4-D Dicamba NAA IBA all synthetic hormones
9.
1957-58 Miller and Skoog University
of Wisconsin - Madison discovered Kinetin
10.
Kinetin a cytokinin plays active
role in organogenesis
11.
1958 Steward developed somatic
embryo from carrot cells
12.
1958-60 Morel cultured orchids
and dahlias freed them from a viral disease
13.
1962 Murashige and Skoog published
recipe for MS Medium
14.
60’s 70’s Murashige
cloned plants in vitro promoted development of commercial plant T.C. labs
15.
1966 raised haploid plants
from pollen grains
16.
1972 used protoplast fusion
to hybridize 2 species of tobacco into one plant contained 4N
17.
4N all chromosomes of
both plants
18.
70’s 80’s develop techniques
to introduce foreign DNA into plant cells beginning of genetic engineering
19.
T.C. Media functions provide H2O provide
mineral nutritional needs
20.
T.C. Media provide growth
regulators Provide vitamins provide organic compounds
21.
T.C. Media provide access
to atmosphere for gas exchange serve as a dumping ground for plant metabolites
22.
T.C. Media H2O is
usually distilled minerals must provide 17 essential elements energy source and carbon skeletons - sucrose is preferred
23.
Vitamins thiamine pyridoxin nicotinic acid biotin
24.
Vitamins citric acid ascorbic acid inositol
25.
Growth Regulators auxins and
cytokinins gibberellic acid abscissic acid
26.
pH of media usually
5.0-5.7
27.
Media must be sterile autoclave
at 250 F at 15 psi for 15 minutes
28.
T.C. Stages Explanting- Stage
I get plant material in sterile culture so it survives provide with nutritional and light needs for growth
29.
Stage II rapid multiplication stabilized
culture goal for a commercial lab difficult and time consuming to maintain
30.
Stage II occurs in
different pathways in different plants
31.
Rooting - Stage
III may occur in Stage II usually induced by changes in hormonal environment lower cytokinin concentration and increase auxin
32.
Rooting may skip stage
III and root in a greenhouse
33.
Stage IV transplantation and
aftercare usually done in greenhouse keep RH high (relative humidity)
34.
Stage IV gradually increase
light intensity and lower RH after rooting occurs allows plants to harden and helps plants form cuticle
35.
Cuticle waxy substance promotes
development of stomates plants in T.C. don’t have cuticle
36.
Explant portion of plant
removed and used for T.C. Important features size source - some tissues are better than others
37.
Explant species dependent physiological age
- young portions of plant are most successful
38.
Explant degree of contamination external
infestation - soak plant in sodium hypochlorite solution
39.
Explant internal infection -
isolate cell that is not infected roots - especially difficult because of soil contact
40.
Explant herbaceous plants soft stem easier
to culture than woody plants
41.
Patterns of
multiplication stage II - light 100-300 foot candles callus - shoots - roots stage III - rooting - light intensity 1000-3000 foot candles
42.
Genetic
transformation permanent incorporation of new or foreigh DNA into genome of cell
43.
Transformation
methods protoplast fusion cell wall is enzymatically removed from cell
44.
Protoplasts naked plant cells from
2 different plants can be mixed together and forced to fuse
45.
Protoplast fusion results in
heterokaryon cell containing two or more nuclei from different cells homokaryon - from same cell
46.
Protoplast fusion allowed to
regenerate cell wall and then grow into callus callus turns to shoots
47.
Shotgun approach DNA coated
micro bullets of gold or tungston shot into growing cells DuPont holds the patent
48.
Shotgun approach injures cells random
success rate
49.
PEG Polyethylene glycol pores open
similar to electroporation
50.
Ti Plasmids Tumor inducing Agrobacterium
temefasciens infect cells with agrobacterium which contains desired DNA
51.
Ti Plasmids monocots resist
agrobacterium infection researchers are working to overcome this
52.
Luciferase an enzyme put into
tobacco using Ti plasmid
53.
Luciferase when transformed tobacco
plants are watered with solution containing Luciferin they break it down and emit light
54.
Luciferase glowing in the
dark like a fire fly
55.
Screening techniques used to
identify if culture has taken on desired new trait
56.
Examples sensitivity to antibiotics color sensitivity
to excess deficiencies of substances in growth media
57.
Conventional plant breeding egg cell
gives half the chromosomes and almost all of the cytoplasm male only gives its chromosomes
58.
Cont……. This condition is
called maternal cytoplasmic inheritance
59.
Microinjection single cells from
culture are held stationary with gentle suction injected with a tiny syringe loaded with DNA
60.
Microinjection done under electron
microscope
61.
Electroporation desired DNA in
solution outside cell high energy pulses - 50,000 volts for a millisecond
62.
Electroporation cause tiny pores
to open allows DNA to enter the cell
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