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

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.

Tobacco Callus

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.

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

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

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. 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

Callus Growth Is Predominantly at the Periphery of the Tissue

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.

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

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

Shoot Organogenesis of Tobacco High cytokinin Low cytokinin

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

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