Plant tissue culture involves growing plant cells, tissues, or organs in a sterile nutrient medium. Key developments included the discovery that single plant cells could be cultured in 1902, the role of growth regulators in the 1930s, and the development of techniques like protoplast fusion and genetic engineering in the 1970s-1980s. Successful tissue culture involves explant preparation, multiplication of cultures, rooting, and transplantation to the greenhouse. Various methods can be used to introduce foreign DNA, including Agrobacterium infection, microinjection, and electroporation.

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