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Plant Cell, Tissue and Organ Culture Hort 515 Callus Cultures 1.Definition and Background 2. Initiation and Establishment of Callus I. Explant II. Nutrient

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  • Plant Cell, Tissue and Organ Culture Hort 515 Callus Cultures 1.Definition and Background 2. Initiation and Establishment of Callus I. Explant II. Nutrient medium III. Temperature and light requirements 3.Callus Maintenance 4.Callus Growth Patterns I. Growth patterns leading to organized development II. Growth patterns leading to continued proliferation of unorganized callus
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  • 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 1. Definition and Background 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.
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  • Tobacco Callus
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  • 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. 1. Definition and Background 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.
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  • 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 andgrowth while actively proliferating cells require only nutrients for continued growth Callus Formation/Proliferation Is Due to:
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  • 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 Background
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  • Plant Cell, Tissue and Organ Culture Hort 515 Callus Cultures 1.Definition and Background 2. Initiation and Establishment of Callus I. Explant II. Nutrient medium III. Temperature and light requirements 3.Callus Maintenance 4.Callus Growth Patterns I. Growth patterns leading to organized development II. Growth patterns leading to continued proliferation of unorganized callus
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  • A.Diversity (genetic) of cell types - less differentiated cells are more responsive to callus induction on media of simple composition, example B.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 2. Initiation and Establishment of Callus I.Explant II.Nutrient medium II.Temperature and light requirements I. Explant
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  • 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 Isolated roots were cultured Auxin and Cytokinin Facilitate the Proliferation of Different Cell Types
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  • A.Diversity (genetic) of cell types - less differentiated cells are more responsive to callus induction on media of simple composition A.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 2. Initiation and Establishment of Callus I. Explant
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  • Jerusalem artichoke tuber explants Months in Storage 0 24 48 72 036912 Time 1 st Cell Division (hours) Storage Increases Time to 1st Cell Division
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  • A.Diversity (genetic) of cell types - less differentiated cells are more responsive to callus induction on media of simple composition A.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 2. Initiation and Establishment of Callus I. Explant
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  • II. Nutrient Medium A.Mineral nutrients - essential micro- and macronutrients B.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: i.Auxin and cytokinin autotrophic tissues - immature lemon fruit, genetic tumor producing plants i.Cytokinin autotrophic - i.e. requires auxin - cereal callus, carrot root i.Auxin autotrophic - i.e. requires cytokinin - turnip root, carrot ii.Auxin and cytokinin auxotrophic - most dicots iii.Auxin and cytokinin auxotrophic, and require complex natural extracts - orchid seedlings
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  • A.Temperature - 24 to 28C B. Light - Dark or diffuse light (l000 lux) 20 E m -2 s - 1 III. Culture Environment
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  • Plant Cell, Tissue and Organ Culture Hort 515 Callus Cultures 1.Definition and Background 2. Initiation and Establishment of Callus I. Explant II. Nutrient medium III. Temperature and light requirements 3.Callus Maintenance 4.Callus Growth Patterns I. Growth patterns leading to organized development II. Growth patterns leading to continued proliferation of unorganized callus
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  • 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
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  • Arabidopsis Tobacco 3.0 mg/L 2,4-D Genotypic Effects on Callus Morphology Compact Callus Friable Callus
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  • Medium Effects on Tobacco Callus Morphology 0.1 mg/L kinetin 3.0 mg/L 2,4-D 2.0 mg/L IAA 3.0 mg/L 2-iP friable callus compact callus
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  • 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 3. Maintenance of Callus
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  • 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 e

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