6. Definition:
The term plant tissue culture is described in vitro and aseptic cultivation of any plant part on nutrient medium. OR
the culture of plant seeds, organs, tissues, cells, or protoplasts on nutrient media under sterile conditions.

7. Factors Affecting Plant Tissue Culture
Growth Media
Minerals, Growth factors, Carbon source, Hormones
Environmental Factors
Light, Temperature, Photoperiod, Sterility, Media
Explant Source
Usually, the younger, less differentiated the explant, the better for tissue culture
Genetics
Different species show differences in amenability to tissue culture
In many cases, different genotypes within a species will have variable responses to tissue culture; response to somatic embryogenesis has been transferred between melon cultivars through sexual hybridization
Emphasize the implications for genetic involvement: Could there be undesirable genes linked to genes influencing tissue culture response?

8. Three Fundamental Abilities of Plants
Totipotency
the potential or inherent capacity of a plant cell to develop into an entire plant if suitably stimulated.
It implies that all the information necessary for growth and reproduction of the organism is contained in the cell
Dedifferentiation
Capacity of mature cells to return to meristematic condition and development of a new growing point, follow by redifferentiation which is the ability to reorganise into new organ
Competency
the endogenous potential of a given cells or tissue to develop in a particular way

9. Types of In Vitro Culture
Culture of intact plants (seed and seedling culture)
Embryo culture (immature embryo culture)
Organ culture
1. shoot tip culture
2. root culture
3. leaf culture
4. anther culture
Callus culture
Cell suspension culture
Protoplast culture

10. Engraving of Henri-Louis Duhamel du Monceau by François-Hubert Drouais
Engraving of Henri-Louis Duhamel du Monceau by François-Hubert Drouais. Duhamel du Monceau was the first to describe callus formation that he observed growing over the wound of an elm tree.

11. Callus formation

12. Theodor Schwann
Theodor Schwann (7 December 1810 – 11 January 1882) was a German physiologist.
His many contributions to biology include the development of cell theory.

13. Performed experiment on callus formation by decorticated trees such as Robinia, Pawlonia and Ulmus.
Trecul (1853)
Obtained very flourishing callus from Brassica rapa and proposed the polarity in development of buds from the upper portion and roots or callus and from the lower portion of a stem piece
Vochting (1878)
Described callus formation on isolated stem fragments and root slices.
Rechinger (1893)
Concept of in vitro Cell Culture
Gottlieb Haberlandt (1902)
(Father of Tissue Culture)
Culture of Embryogenic tissue
Hannig (1904)

14. Gottlieb Haberlandt
Haberlandt first pointed out the possibilities of the culture of isolated tissues, plant tissue culture.
His original idea presented in 1902 was called totipotentiality: “Theoretically all plant cells are able to give rise to a complete plant.”

15. *All these discoveries contributed to the establishment of totipotency of somatic cells under experimental conditions, thereby accomplishing the goals set by Haberlandt.

16. History
In 1902 Haberlandt proposed that single plant cells could be cultured

17. Haberlandt
did not culture them himself

18. 1930’s
White worked on T.C.
discovery of plant growth regulators

19. 1930’s
importance of vitamins was determined for shoot and root culturing
A,D,E,K,C, B complex

20. 1930’s
Indole-Acetic Acid
IAA
discovered in 1937

21. IAA
2,4-D
Dicamba
NAA
IBA
all synthetic hormones

22. 1957-58 Miller and Skoog University of Wisconsin - Madison
discovered Kinetin

23. Kinetin
a cytokinin
plays active role in organogenesis

24. 1958
Steward developed somatic embryo from carrot cells

25. 1958-60 Morel cultured orchids and dahlias
freed them from a viral disease

26. 1962
Murashige and Skoog
published recipe for MS Medium

27. 60’s & 70’s Murashige cloned plants in vitro
promoted development of commercial plant T.C. labs

28. 1966
raised haploid plants from pollen grains

29. 1972
used protoplast fusion to hybridize 2 species of tobacco into one plant
contained 4N

30. 4N
all chromosomes of both plants

31. 70’s &80’s
develop techniques to introduce foreign DNA into plant cells
beginning of genetic engineering

32. HISTORY OF PLANT TISSUE CULTURE
cellular theory (Cell is autonom and totipotent)
Schleiden-Schwann
1902
First attempt of plant tissue culture
Harberlandt
1939
Continuously growing callus culture
White
1946
Whole plant developed from shoot tip
Ball
1950
Organs regenerated on callus
1954
Plant from single cell
Muir
1960
Protoplast isolation
Cocking

33. HISTORY OF PLANT TISSUE CULTURE
1962
MS media
Murashige - Skoog
1964
Clonal propagation of orchids
Morel
Haploids from pollen
Guha
1970
Fusion of protoplasts
Power
1971
Plants from protoplasts
Takebe
1981
Somaclonal variation
Larkin

34. Development of Tissue Culture
Root-tip Culture
White (1934, 1937)
Embryo Culture
(Use of coconut milk i.e. embryo sac fluid)
Van Overbeek et al. (1941)
Stem-tip Culture
Loo (1945)
Ball (1946)
Role of Auxin
Cambium cells of some tree species
Gautheret (1939)
Tumor tissues of hybrid
White (1939)
Carrot slices
Nobecourt (1939)
Discovery of Cytokinin
Skoog (1955)
Miller et al. (1955)

35. Hormonal Control of Organ Formation
Skoog and Miller (1957)
Improvement of Media
Murashige and Skoog (1962)
Preparation and Cloning of Single Cell Cultures
Bergmann (1960)
Regeneration of Single Cell to Whole Plant
Regenerated plantlets
Vasil (1965)
Somatic embryogenesis
Steward (1966)
Kohlenbach (1966)

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

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

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

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

40. Vitamins
thiamine
pyridoxin
nicotinic acid
biotin

41. Vitamins
citric acid
ascorbic acid
inositol

42. Growth Regulators auxins and cytokinins gibberellic acid
abscissic acid

43. Basis for Plant Tissue Culture
Two Hormones Affect Plant Differentiation:
Auxin: Stimulates Root Development
Cytokinin: Stimulates Shoot Development
Generally, the ratio of these two hormones can determine plant development:
 Auxin ↓Cytokinin = Root Development
 Cytokinin ↓Auxin = Shoot Development
Auxin = Cytokinin = Callus Development

44. Control of in vitro culture
Cytokinin
Leaf strip
Adventitious
Shoot
Root
Callus
Auxin

45. pH of media
usually

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

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

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

49. Stage II
occurs in different pathways in different plants

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

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

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

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

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

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

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

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

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

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

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

61. Genetic transformation
permanent incorporation of new or foreigh DNA into genome of cell

62. Transformation methods
protoplast fusion
cell wall is enzymatically removed from cell

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

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

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

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

67. Shotgun approach
injures cells
random success rate

68. PEG
Polyethylene glycol
pores open similar to electroporation

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

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

71. Luciferase
an enzyme
put into tobacco using Ti plasmid

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

73. Luciferase
glowing in the dark
like a fire fly

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

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

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

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

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

79. Microinjection
done under electron microscope

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

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

82. Fundamental principles:
There are three fundamental principles
(1) The plant part must be isolated from the rest of plant body.
(2) The plant part (ex plant; isolate cell that is not infected) must be in a controlled , preferably defined , nutrient medium.
(3) Aseptic condition must be maintained.

83. Types of plant cultures:
(1) Callus Culture.
(2) Cell Suspension Culture.
(3) Anther Culture.
(4) Ovule Culture.
(5) Embryo Culture.
(6) Protoplast Culture.
(7) Micro Propagation.

84. History: (1) HABERLANDT. (2) WHITE. (3) NOBECOURT. (4) VAN OVERBEEK.
(5) SKOOG.
(6) BRAUN.
(7) STEWARD.
(8) MURI.

85. (1) Callus Culture technique
When an excised and isolated piece of tissue is cultured on a nutrient medium, an unorganized mass of cell appears, is called callus. This callus is transferred on to different media to regenerate plants. this technique is called tissue culture.

86. Plant tissue culture

87. (2) Cell Suspension Culture
It is the culture of isolated cells or very small cell aggregates dispersed in liquid medium. The cell suspension is obtained by agitating pieces of callus in liquid medium on gyrating shaker.

88. (3) Anther Culture
Anther culture is a mean to produce plants with a gametic number of chromosomes by aseptic culture of anther. The technique give rise to haploid plants either directly or through formation of a haploid callus.

89. (4) Ovule Culture
Ovule culture techniques is an important technique in modern plant breeding. it is much easier to culture whole ovule than to isolate a single embryo, especially in small seeded plants.
E.g., tobacco

90. (5) Embryo Culture
The embryos are isolated from young seeds and placed on a solid medium containing nutrients and vitamins. Embryos are cultured at 25°c,first in dark until seedlings are about 2 cm long and root formation has started, and than in light until the seedlings can be planted in soil.

91. (6) Protoplast Culture
It is one of the most significant and recent developments in the field of plant tissue culture. the protoplast are usually isolated from cultured cell or leaf mesophyll cell by treating them with enzyme solutions. the isolated protoplast may be used to regenerate the plants directly, or for the production of somatic hybrids through fusion.

92. (7) Micro Propagation
It is in vitro asexual propagation of crop plants. This technique is advantages over the conventional practice of asexual propagation as only a small amount of plant is needed, species highly resistant to conventional bulk propagation can be propagated by this method and it is non season dependent. Micro propagation is used for rapid multiplication of stocks, elimination of diseases, germ plasm preservation, and induction of mutation.

93. Applications:
Tissue culture technique has helped in studying various biochemical and physiological processes in pure cultures without complications of other factors.
The effect of various hormones are differentiation and growth processes can be studied by using tissue culture technique.
Organogenesis has been studied by inducing formation of roots , shoot tips etc from callus on nutrient medium.