1. Plant Cell, Tissue and Organ Culture
Hort 515
Callus Cultures
Definition and Background
2. Initiation and Establishment of Callus
I. Explant
II. Nutrient medium
III. Temperature and light requirements
Callus Maintenance
Callus Growth Patterns
I. Growth patterns leading to organized development
II. Growth patterns leading to continued proliferation of unorganized callus

2. 1. Definition and Background
Callus – A tissue that develops in response to injury caused by physical or chemical means
Most callus cells are differentiated although may be and are often highly unorganized within the tissue
Most common form of callus is the wound tissue that produces a protective layer of cells to cover an injury
Callus culture example
Differentiated Cells - products of cell differentiation, i.e. specific cell types with particular function, e.g. xlyem tracheary elements
Cells after expansion large cells with prominent vacuoles and little cytoplasm
Undifferentiated Cells - meristematic; progenitors of differentiated somatic cells, e.g. shoot and root apices, small, isodiametric, small vacuoles.

3. Tobacco Callus

4. 1. Definition and Background
Callus – A tissue that develops in response to injury caused by physical or chemical means, most cells of which are differentiated although may be and are often highly unorganized within the tissue. In nature, this wound tissue produces a protective layer of cells to cover an injury, example.
Differentiated Cells - products of cellular maturation, i.e. cell types with particular function, e.g. xylem tracheary elements; large cells that are highly vacuolated with relatively little cytoplasm
Undifferentiated Cells - meristematic; progenitors of differentiated somatic cells, e.g. small, isodiametric, small vacuoles.

5. Callus Formation/Proliferation Is Due to:
Removal of cells within the explant from organizational controls (genetic/chemical) inter-cellular, -tissue and –organ “cross- talk” that programs morphological development
Cells are “released” from organizational controls that are exerted by other cells as part of the developmental program
Provision of mineral nutrients and growth regulators for autonomous and indeterminate cell growth
Highly differentiated (quiescent) cells require stimuli (e.g. growth regulators) for cell division induction and growth while actively proliferating cells require only nutrients for continued growth

6. Background
Haberlandt (1902) - Hypothesized the existence of auxins and cytokinins based on callus formation after wounding of potato tuber pieces. Production of potato “seed” involves a finite number of divisions.
Haberlandt predicted that “division” and growth factors (expansion) facilitate indeterminate growth and totipotency, i.e. formation of new plants (somatic embryogenesis)
cytokinins – cell division, auxins – cell expansion
Kogel, Hagen-Smit and Thimann (mid 1930s) - discovered auxin
First Callus Cultures (1939): Plant cells are capable of indeterminate growth, prelude to totipotency
Gautheret and Nobecourt - callus from carrot roots, medium containing auxin (cytokinin autotrophic)
White - Nicotiana glauca x N. langsdorffii, hybrid naturally forms tumors, hormone autotrophic

7. Plant Cell, Tissue and Organ Culture
Hort 515
Callus Cultures
Definition and Background
2. Initiation and Establishment of Callus
I. Explant
II. Nutrient medium
III. Temperature and light requirements
Callus Maintenance
Callus Growth Patterns
I. Growth patterns leading to organized development
II. Growth patterns leading to continued proliferation of unorganized callus

8. 2. Initiation and Establishment of Callus
Explant
Nutrient medium
Temperature and light requirements
I. Explant
Diversity (genetic) of cell types - less differentiated cells are more responsive to callus induction on media of simple composition, example
Physiological status of the explant – callus induction from the explant will be affected by physiological status, e.g. nutrient status, hormonal content, dormancy status, etc.
C. Genotype - e.g. soybean varieties vary in their requirement for cytokinins, i.e. there are cytokinin autotrophs and auxotrophs

9. Auxin and Cytokinin Facilitate the Proliferation of Different Cell Types
Isolated roots were
cultured
Pea roots contain cells of different ploidy levels; 2n, 4n, 8n, etc. Roots were induced to form callus on either of the following media:
2,4-D and kinetin – 4n cells predominated after one week
2,4-D w/o kinetin – 2n cells predominated after one week
4n cells require cytokinin for division/growth

10. 2. Initiation and Establishment of Callus
I. Explant
Diversity (genetic) of cell types - less differentiated cells are more responsive to callus induction on media of simple composition
Physiological status of the explant – callus induction from explants will be affected by the physiological status of the plant, e.g. nutrient status, hormonal content, dormancy status, etc., example
C. Genotype - e.g. soybean varieties vary in their requirement for cytokinins, i.e. there are cytokinin autotrophs and auxotrophs

11. Storage Increases Time to 1st Cell Division
Jerusalem artichoke tuber explants 72
Time
1st Cell
Division
(hours) 48 24 3 6 9 12
Months in Storage

12. 2. Initiation and Establishment of Callus
I. Explant
Diversity (genetic) of cell types - less differentiated cells are more responsive to callus induction on media of simple composition
Physiological status of the explant – callus induction from explants will be affected by the physiological status of the plant, e.g. nutrient status, hormonal content, dormancy status, etc., example
C. Genotype - e.g. soybean varieties vary in their requirement for cytokinins, i.e. there are cytokinin autotrophs and auxotrophs

13. II. Nutrient Medium
Mineral nutrients - essential micro- and macronutrients
Organic constituents – “basal” constituents are sucrose or glucose/fructose as carbon sources and usually I-inositol and thiamine-HCl. Five basic groups of callus tissue types based on growth regulator requirements:
Auxin and cytokinin autotrophic tissues - immature lemon fruit, genetic tumor producing plants
Cytokinin autotrophic - i.e. requires auxin - cereal callus, carrot root
Auxin autotrophic - i.e. requires cytokinin - turnip root, carrot
Auxin and cytokinin auxotrophic - most dicots
Auxin and cytokinin auxotrophic, and require complex natural extracts - orchid seedlings

14. III. Culture Environment
Temperature - 24 to 28°C
B. Light - Dark or diffuse light (l000 lux) 20 E m-2 s-1

15. Plant Cell, Tissue and Organ Culture
Hort 515
Callus Cultures
Definition and Background
2. Initiation and Establishment of Callus
I. Explant
II. Nutrient medium
III. Temperature and light requirements
Callus Maintenance
Callus Growth Patterns
I. Growth patterns leading to organized development
II. Growth patterns leading to continued proliferation of unorganized callus

16. General - Callus induction and maintenance media contain the same basal constituents with the exception that most callus requires auxin and cytokinin (auxotrophic) in the maintenance medium, particularly after prolonged culture (except habituated cells).
Callus is re-cultured after 4 to 6 cell doublings, when growth becomes nutrient limited in a batch culture. This interval is referred to as a passage.
Callus morphology - Callus differs in compactness or looseness, i.e. cells may be tightly joined and the tissue mass is one solid piece or cells are loosely joined and individual cells readily separable (friable), which is affected by the genotype or the medium composition, examples
A friable callus is often used to initiate a liquid cell suspension culture
3. Maintenance of Callus

17. Genotypic Effects on Callus Morphology
Arabidopsis Tobacco
3.0 mg/L 2,4-D
Compact Callus
Friable Callus

18. Medium Effects on Tobacco Callus Morphology
2.0 mg/L IAA
3.0 mg/L 2-iP
0.1 mg/L kinetin
3.0 mg/L 2,4-D
friable callus
compact callus

19. 3. Maintenance of Callus
General - Callus induction and maintenance media contain the same basal constituents with the exception that most callus requires auxin and cytokinin (auxotrophic) in the maintenance media, particularly after prolonged culture (except habituated cells).
Callus is re-cultured after 4 to 6 cell doublings, when growth becomes nutrient limited in a batch culture. This interval is referred to as a passage.
Callus morphology - Callus differs in compactness or looseness, i.e. cells may be tightly joined and the tissue mass is one solid piece or cells are loosely joined and individual cells readily separate (friable), and is affected by the genotype or the medium composition, examples
Friable callus is often used to initiate a liquid cell suspension culture

20. Cytogenetic/genetic variation - Cells of callus are genetically very heterogeneous and the heterogeneity increases during culture
Regenerated plants will reflect this genetic variation (somaclonal variation). However, morphogenetic competence is more associated with genetically stable (e.g. meristematic) cells
The cytogenetic changes that occur are polyploidy/aneuploidy, translocation, amplification, methylation, epigenetics etc, although the exact genetic basis for most somaclonal variation is unknown
Cytogenetic variation can be minimized by choosing explants that are meristematic and maintain callus in media that favor cell division
Somaclonal variation – genetic variation that arises in somatic (non-germ line) cells
3. Maintenance of Callus

21. Plant Cell, Tissue and Organ Culture
Hort 515
Callus Cultures
Definition and Background
2. Initiation and Establishment of Callus
I. Explant
II. Nutrient medium
III. Temperature and light requirements
Callus Maintenance
Callus Growth Patterns
I. Growth patterns leading to organized development
II. Growth patterns leading to continued proliferation of unorganized callus

22. 4. Callus Growth Patterns
Growth patterns leading to organized development - morphogenesis (adventitious organogenesis or somatic embryogenesis)
Callus growth is quantified measurements of fresh or dry weight, cell number, cell volume, mitotic index (% of cells in mitosis) and DNA content
Growth patterns leading to continued proliferation of unorganized callus – maintenance

23. I. Growth patterns leading to organized development
Induction of growth (manifested as a lag) - Fresh medium induces quiescent cells (stationary phase) to enter the cell cycle, G1SG2M.
Cells in G1 phase proceed through S (DNA/RNA synthesis) phase and then through a short G2 phase prior to mitosis
Division phase - rapid increase in cell number through periclinal (parallel to nearest surface) divisions subjacent to the periphery of the callus, and followed by anticlinal (perpendicular) divisions, example
Division  fresh weight gain resulting in substantial reduction in cell volume (regressive growth), cells dedifferentiate (become meristematic-like),
C. Differentiation - cell division slows, during this period differentiation occurs which is then followed by cell expansion resulting in the development of an organized structure.

24. Callus Growth Is Predominantly at the Periphery of the Tissue

25. I. Growth patterns leading to organized development
Induction of growth (manifested as a lag) - Fresh medium induces quiescent cells (stationary phase) to enter the cell cycle, G1SG2M.
Cells in G1 phase proceed through S (DNA/RNA synthesis) phase and then through a short G2 phase prior to mitosis
Division phase - rapid increase in cell number through periclinal (parallel to nearest surface) divisions subjacent to the periphery of the callus, and followed by anticlinal (perpendicular) divisions,
Division  fresh weight gain resulting in substantial reduction in cell volume (regressive growth), cells dedifferentiate (become meristematic-like), example
C. Differentiation - cell division slows, during this period differentiation occurs which is then followed by cell expansion resulting in the development of an organized structure.

26. Jerusalem Artichoke Tuber Callus
Cell number increases 10-fold in the first 7 days and cells dedifferentiate into meristematic cells
Phase of regressive
change/
dedifferentiation

27. I. Growth patterns leading to organized development - morphogenesis (adventitious organogenesis or somatic embryogenesis)
Induction of growth (manifested as a lag) - Transfer to fresh medium induces differentiated cells (quiescent) to enter an active cell cycle, i.e. cell division machinery is activated, G1SG2M. Cells are in G1 phase but begin S (DNA/RNA synthesis) and proceed through a short G2 phase prior to mitosis.
Division phase - rapid increase in cell number through periclinal (parallel to nearest surface) divisions at the subjacent to the periphery of the callus, division  fresh weight gain resulting in substantial reduction in cell volume (regressive growth), cells dedifferentiate (become meristematic-like).
C. Differentiation - cell division slows, during this period differentiation occurs which is then followed by cell expansion resulting in the development of an organized structure, examples

28. Shoot Organogenesis of Tobacco
High cytokinin
Low cytokinin

29. Somatic Embryogenesis of Carrot
2,4-D (mg/L)

30. II. Growth Patterns Leading to Continued Proliferation of Unorganized Callus
Induction phase – lag/conditioning
Cell division phase - regressive change but no dedifferentiation
C. Cell expansion - no differentiation