Plant Cell, Tissue and Organ Culture

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 Undiffere
ntiated 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
0
0
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 28C
• 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
Friable Callus
Compact 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
compact callus
friable 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