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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

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slide1

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
slide2

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.

slide4

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.

slide5

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

slide6

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

slide7

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
slide8

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
slide9

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

slide10

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
slide11

Storage Increases Time to 1st Cell Division

Jerusalem artichoke tuber explants

72

Time

1st Cell

Division

(hours)

48

24

0

0

3

6

9

12

Months in Storage

slide12

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
slide13

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
slide14

III. Culture Environment

  • Temperature - 24 to 28°C
  • B.Light - Dark or diffuse light (l000 lux) 20 E m-2 s-1
slide15

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
slide16

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

slide17

Genotypic Effects on Callus Morphology

ArabidopsisTobacco

3.0 mg/L 2,4-D

Friable Callus

Compact Callus

slide18

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

compact callus

friable callus

slide19

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

slide20

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

slide21

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
slide22

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
slide23

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.
slide25

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.
slide26

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

slide27

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
slide28

Shoot Organogenesis of Tobacco

High cytokinin

Low cytokinin

slide30

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