1. Plant Tissue Culture
Matt Jakubik

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
A, D, E, K, C, and B Complex

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
all chromosomes of both plants

17. 70’s &80’s
develop techniques to introduce foreign DNA into plant cells
beginning of genetic engineering

18. T.C. Media
functions
provide H2O
provide mineral nutritional needs

19. T.C. Media provide growth regulators Provide vitamins
provide organic compounds

20. T.C. Media provide access to atmosphere for gas exchange
serve as a dumping ground for plant metabolites

21. T.C. Media H2O is usually distilled
minerals must provide 17 essential elements
energy source and carbon skeletons - sucrose is preferred

22. Vitamins
thiamine
pyridoxin
nicotinic acid
biotin

23. Vitamins
citric acid
ascorbic acid
inositol

24. Growth Regulators auxins and cytokinins gibberellic acid
abscissic acid

25. pH of media
usually

26. Media
must be sterile
autoclave at 250 F at 15 psi for 15 minutes

27. T.C. Stages Explanting- Stage I
get plant material in sterile culture so it survives
provide with nutritional and light needs for growth

28. Stage II rapid multiplication stabilized culture
goal for a commercial lab
difficult and time consuming to maintain

29. Stage II
occurs in different pathways in different plants

30. Rooting - Stage III may occur in Stage II
usually induced by changes in hormonal environment
lower cytokinin concentration and increase auxin

31. Rooting
may skip stage III and root in a greenhouse

32. Stage IV transplantation and aftercare usually done in greenhouse
keep RH high (relative humidity)

33. Stage IV
gradually increase light intensity and lower RH after rooting occurs
allows plants to harden and helps plants form cuticle

34. Cuticle waxy substance promotes development of stomates
plants in T.C. don’t have cuticle

35. Explant portion of plant removed and used for T.C. Important features
size
source - some tissues are better than others

36. Explant species dependent
physiological age - young portions of plant are most successful

37. Explant degree of contamination
external infestation - soak plant in sodium hypochlorite solution

38. Explant internal infection - isolate cell that is not infected
roots - especially difficult because of soil contact

39. Explant herbaceous plants soft stem
easier to culture than woody plants

40. Patterns of multiplication
stage II - light foot candles
callus - shoots - roots
stage III - rooting - light intensity foot candles

41. Genetic transformation
permanent incorporation of new or foreign DNA into genome of cell

42. Transformation methods
protoplast fusion
cell wall is removed by enzymes from cell

43. Protoplasts naked plant cells
from 2 different plants can be mixed together and forced to fuse

44. Protoplast fusion results in heterokaryon
cell containing two or more nuclei from different cells
homokaryon - from same cell

45. Protoplast fusion
allowed to regenerate cell wall and then grow into callus
callus turns to shoots

46. Shotgun approach DNA coated micro bullets of gold or tungston
shot into growing cells
DuPont holds the patent

47. Shotgun approach
injures cells
random success rate

48. PEG
Polyethylene glycol
pores open similar to electroporation

49. Ti Plasmids Tumor inducing Agrobacterium temefasciens
infect cells with agrobacterium which contains desired DNA

50. Ti Plasmids monocots resist agrobacterium infection
researchers are working to overcome this

51. Luciferase
an enzyme
put into tobacco using Ti plasmid

52. Luciferase
when transformed tobacco plants are watered with solution containing Luciferin
they break it down and emit light

53. Luciferase
glowing in the dark
like a fire fly

54. Screening techniques
used to identify if culture has taken on desired new trait

55. Examples sensitivity to antibiotics color
sensitivity to excess deficiencies of substances in growth media

56. Conventional plant breeding
egg cell gives half the chromosomes and almost all of the cytoplasm
male only gives its chromosomes

57. Cont…….
This condition is called maternal cytoplasmic inheritance

58. Microinjection
single cells from culture are held stationary with gentle suction
injected with a tiny syringe loaded with DNA

59. Microinjection
done under electron microscope

60. Electroporation desired DNA in solution outside cell
high energy pulses - 50,000 volts
for a millisecond

61. Electroporation
cause tiny pores to open
allows DNA to enter the cell