Definitions and Background Information

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Plant Cell, Tissue and Organ Culture Hort
515 Plant Regeneration

• Definitions and Background Information
• Morphogenetic Processes That Lead to Plant
  Regeneration
• Factors Affecting Morphogenesis in Vitro
• Genetic Variation of Plants Regenerated in Vitro
• Regeneration of Cereals

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1. Definitions and Background Information
Plant Morphology - form and structure of
plants Plant Morphogenesis - differentiation and
formation of organized structures specifically
processes that lead to plant regeneration from
somatic cells, i.e., formation of shoots and
roots (organogenesis) or somatic/adventive/asexual
embryos (embryogenesis) Somatic cells - cells
of the plant body, not gametes Regenerated
plants are clones, within the limits of somatic
cell variation, are exact genetic copies of the
parent Genetic variation of regenerated plants
is dependent on genetic constitution of somatic
cells in the explant, and degree to which this
variation increases in culture prior to
regeneration.
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2. Morphogenetic Processes that Lead to Plant
Regeneration

• I. Organogenesis formation of organs, i.e.
  shoots and roots
• Enhancement of axillary bud proliferation/
  development
• Adventitious shoot formation
• Adventitious root formation
• II. Somatic (asexual/adventive) embryogenesis

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• Organogenesis - shoot initiation and development
  with subsequent formation of adventitious roots
• Adventitious - initiation from cells that are
  not normally the progenitors, e.g.
  non-meristematic cells

• Enhancement of axillary bud proliferation and
  development - stimulation of the shoot apical
  meristem differentiation and development,
  proliferation of lateral buds to shoots, example
• Adventitious shoot formation - dedifferentiation
  and/or differentiation and development of shoots
  from nonmeristematic cells (one or more than one)
  either

• Direct - cells of explant dedifferentiate
  (meristemoids) and/or then differentiate into
  adventitious shoots
• Indirect - callus is proliferated from the
  primary explant, dedifferentiates into
  meristemoids and then differentiate into shoots

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Shoot Tip Propagation of Asparagus by Enhancement
of Axillary Bud Development
Six (6) shoots each passage Six (6)
passages/year 46656 plants/yr
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• Organogenesis - shoot initiation and development
  with subsequent formation of adventitious roots
• Adventitious - initiation from cells that are
  not normally the progenitors, e.g.
  non-meristematic cells

• Enhancement of axillary bud proliferation and
  development - stimulation of the shoot apical
  meristem differentiation and development,
  proliferation of lateral buds to shoots, example
• Adventitious shoot formation - dedifferentiation
  and/or differentiation and development of shoots
  from nonmeristematic cells (one or more than one)
  either

• Direct - cells of explant dedifferentiate
  (meristemoids) and/or then differentiate into
  adventitious shoots, example
• Indirect - callus is proliferated from the
  primary explant, dedifferentiates into
  meristemoids and then differentiate into shoots

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Direct Adventitious Shoot Bud Development in
Culture from Douglas Fir Cotyledon Explants
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• Organogenesis - shoot initiation and development
  with subsequent formation of adventitious roots
• Adventitious - initiation from cells that are
  not normally the progenitors, e.g.
  non-meristematic cells

• Enhancement of axillary bud proliferation and
  development - stimulation of the shoot apical
  meristem differentiation and development,
  proliferation of lateral buds to shoots, example
• Adventitious shoot formation - dedifferentiation
  and/or differentiation and development of shoots
  from nonmeristematic cells (one or more than one)
  either

• Direct - cells of explant dedifferentiate
  (meristemoids) and/or then differentiate into
  adventitious shoots, example
• Indirect - callus is proliferated from the
  primary explant, dedifferentiates into
  meristemoids and then differentiate into shoots

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Tobacco Meristemoid Prior to Morphogenesis
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Meristmoids - can give rise to several shoots, so
those arising from one meristemoid may be clones,
also meristmoids may derive from more than one
cell leading to chimerism, example
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Shoot Meristems Differentiated from an Individual
Meristemoid of Potato Tuber Explants
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I. Organogenesis - shoot initiation and
development with subsequent formation of
adventitious roots adventitious - initiation
from cells that are not normally the progenitors,
e.g. non-meristematic cells

• Enhancement of axillary bud proliferation and
  development - stimulation of the shoot apical
  meristem in vitro that includes proliferation of
  lateral buds
• Adventitious shoot formation - dedifferentiation
  and/or differentiation and development of shoots
  from non-meristematic cells (one or more than
  one) either directly or indirectly
• Adventitious root formation - roots are initiated
  adventitiously at the base of the shoot apex and
  a vascular continuum is established to complete
  plant regeneration, example

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Adventitious Root Initiation in an Asparagus
Shoot Tip
Adventitious Root Initiation in a Rose Shoot
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• Somatic embryogenesis - embryo initiation and
  development from somatic cells
• Directly from cells in the explant, examples
• Indirectly via a callus intermediary
  dedifferentiation is typically minimal but a
  meristemoid-like tissue can be formed in the
  latter case
• Histogenesis of somatic embryogenesis is
  characterized by the formation of a bipolar
  structure, in contrast to adventitious
  organogenesis
• Single cell origin of somatic embryos makes
  chimerism infrequent adventitious shoots can
  arise from more than one cell

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Direct Embryogenesis from Cacao Cotyledon
Epidermal Cell
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Somatic Embryogenesis from Cacao Cotyledons
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• Somatic embryogenesis - embryo initiation and
  development from somatic cells
• Directly from cells in the explant
• Indirectly via a callus intermediary
  dedifferentiation is typically minimal but a
  meristemoid-like tissue can be formed in the
  latter case, examples
• Histogenesis of somatic embryogenesis is
  characterized by the formation of a bipolar
  structure, in contrast to adventitious
  organogenesis
• Single cell origin of somatic embryos makes
  chimerism infrequent adventitious shoots can
  arise from more than one cell

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Indirect Somatic Embryogenesis from Cacao
Cotyledons
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• Somatic embryogenesis - embryo initiation and
  development from somatic cells
• Directly from cells in the explant
• Indirectly via a callus intermediary
  dedifferentiation is typically minimal but a
  meristemoid-like tissue can be formed in the
  latter case
• Histogenesis of somatic embryogenesis is
  characterized by the formation of a bipolar
  structure, in contrast to adventitious
  organogenesis, example
• Single cell origin of somatic embryos makes
  chimerism infrequent adventitious shoots can
  arise from more than one cell

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Somatic Embryos of Cacao Illustrating Bipolar
Meristematic Structures
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• Somatic embryogenesis - embryo initiation and
  development from somatic cells
• Directly from cells in the explant
• Indirectly via a callus intermediary
  dedifferentiation is typically minimal but a
  meristemoid-like tissue can be formed in the
  latter case
• Histogenesis of somatic embryogenesis is
  characterized by the formation of a bipolar
  structure, in contrast to adventitious
  organogenesis
• Single cell origin of somatic embryos makes
  chimerism infrequent adventitious shoots can
  arise from more than one cell

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3. Factors Affecting Morphogenesis in Vitro

• I. Explant and explant source
• II. Culture medium
• III. Culture environment
• I. Explant and explant source
• Totipotency, competence, determination and
  development
• Cell and tissue type
• Explant source age
• Genotype
• Genes that regulate morphogenesis leading to
  plant regeneration

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Totipotency, competence, determination and
development cellular differentiation and
developmental states in morphogenesis
Totipotency - genetic potential of cells for
embryogenesis now extended to include
organogenesis, as proposed by Haberlandt
Ground state cell steady state, cells may
be competent (meristematic or meristematic-like)
or may be incompetent Competence competent
cells are undifferentiated and retain the
capacity for differentiation and morphogenesis
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Totipotency, competence, determination and
development cellular differentiation and
developmental stages of morphogenesis
Dedifferentiation cells re-acquire
morphogenetic competence Determination -
competent cells become committed to a genetic
program leading to morphogenesis induced - in
response to a stimulus or permissive - preset
determination is allowed to proceed Development
developmental pathway is fixed, i.e.
organogenesis or embryogenesis Example -
diagramatic example of cellular stages
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Topics in Biotechnology/Plant Biology Lozovaya
et al. (2006) Biochemical features of maize
tissues with different capacities to regeneration
plants. Planta 2241385-1399 Vidi et al.
(2007) Pastoglobules a new address for
targeting recombinant proteins in the
chloroplast. 74
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Cell Developmental Phases Leading to Morphogenesis
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Certain cell types retain competence
regeneration practice dictates isolation of these
for induced or permissive determination Cells
may be induced to re-acquire competence by
differentiation but this has been demonstrated
for only a few genotypes, e.g. tobacco, carrot
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Cell and tissue type - Explants for plant
regeneration are composed of undifferentiated
(meristematic) or differentiated, and
morphogenetically competent or incompetent
cells Explants must contain competent cells or
cells capable of regaining competence
(dedifferentiation)

• Axillary bud proliferation - shoot tip or shoot
  meristem or nodal explants
• Regeneration is based on facilitating
  differentiation and development leading to
  formation and development of axillary buds into
  shoots
• Cells of these explants are competent and
  committed to shoot development

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Cell and tissue type
Axillary bud proliferation Adventitious shoot
formation or somatic embryogenesis Less
differentiated cells tend to be morphogenetically
competent Morphogenetic competence is
associated with meiosis and its
natural expression is during zygotic
embryogenesis/embryogeny Somatic cells
(maternal) associated with meiotic events, e.g.
mircro- and mega-sporogenesis, and micro- and
mega-gametogenesis, are a source of
morphogenetically competent cells Hence,
inflorescence, anther, ovule and ovary tissues,
and embryos are sources of competent
cells Presumably, expression of competence is
repressed during embryogeny and de-repressed
during meiosis, example
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Embryogenic Competence of cells in citrus
nucellar tissue
Citrus varieties are mono-embryonic (zygotic
embryo only) or poly-embryonic (zygotic and
somatic embryos) Somatic embryos are produced
from cells of the nucellus, inner ovular
(maternal cells) tissue Embryo production from
cultured nucellar tissue Variety Intact Micr
opylar half Chalazal half Monoembryonic mono poly
none Polyembryonic poly poly none These results
indicate that cells in the micro-pylar half of
the nucellus are competent for somatic
embryogenesis in both monoembryonic and
polyembryonic citrus types
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Chalazal half of the nucellus from monoembryonic
varieties Inhibits embryogeny of carrot callus

Chalazal half of the citrus nucellus produces
water soluble inhibitors of somatic
embryogenesis
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Citrus/carrot example illustrates
that Competence of cells in tissues is
genetically determined Maternal cells
associated with meiosis may be competent for
embryogenesis Repression of totipotency/competen
ce occurs during embryogenesis and embryogeny and
inhibitors may regulate chemical and/or genetic
repression
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3. Factors Affecting Morphogenesis in Vitro
I. Explant and explant source II. Culture
medium III. Culture environment I. Explant and
explant source A. Totipotency, competence and
determination B. Cell and tissue
type C. Explant source age D. Genotype E. Ge
nes that regulate morphogenesis leading to plant
regeneration
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• Explant source age developmentally immature
  organs are most likely to contain
  morphogenetically competent cells, i.e. less
  differentiated cells are more competent
• Within an organ, loss of competence is correlated
  with maturation, i.e. extent of differentiation,
  examples

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Developmental Stage and Adventitious Shoot
Formation from Tomato Leaf Discs
Fig. 2 Changes in the shoot-forming capacity of
the genotype PU 76-02 associated with changes in
the developmental state of the explant source.
The bars around each mean represent standard
error values. A. Leaves were sampled in
succession from the shoot apex, beginning with
the 1st leaf from the apex at least 5 cm in
length and continuing through the 5th consecutive
leaf. Each point is an average number for 20
explants. B. The 2nd and 3rd leaves numbered in
relation to the apical leaf closest to 5 cm in
length were sampled from plants 6, 8, and 11
weeks after germination. Each point is an average
number for 40 explants. C. Discs were
excised from 3 positions on the leaflet proximal
to the petiole, midleaflet, and distal to the
petiole. Each point is an average number for 30
explants. Z. Pflanzenphysiol. Bd. 102. S.
221-232. 1981.
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Developmental Stage Positions in a Tomato Leaf
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• Genotype - Genotypes can be classified based on
  whether or not cells can be induced in vitro to
  reacquire competence, e.g. carrot/tobacco/potato
  vs. cereals/legumes
• Competent cells must be included in the cultured
  explant of species for which re-acquisition of
  competence cannot be induced
• Morphogenesis in vitro is an inherited trait,
  example

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Adventitious Shoot Forming Capacity of Tomato
Genotypes
?
?
?
?
?
?
?
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Adventitious Shoot Forming Capacity Is Inherited
in Tomato
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Adventitious Shoot Forming Capacity of Tomato Is
Quantitatively Inherited
significant at the 1 level.
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• Genes that regulate plant regeneration
  identification of genes that regulate plant
  development may result in new approaches for
  plant regeneration in vitro
• Adventitious Shoot Formation
• KNAT1 family transcription factors, example
• Cytokinin biosynthetic and signaling genes, etc.
• Somatic Embryogenesis
• SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)
• Transcription factors WUSCHEL, BABYBOOM, LEAFY
  COTYLEDON 1/2, DORNROSCHEN/ENHANCER OF SHOOT
  REGERATION1

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KNAT1 family transcription factors Homeotic
selector genes encode a conserved family of
gene regulatory proteins (transcription factors)
that activate developmental programs in higher
eukaryotes Homeodomain proteins transcription
factors that contain conserved DNA-binding
domains, which interact with cis elements in
promoters of genes necessary for developmental
programs, e.g. vegetative and floral meristem
development
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KNAT1 - homeotic gene involved in shoot
meristem differentiation in Arabidopsis Homeotic
genes were first discovered in Drosophila, are
conserved in higher eukaryotes and orthologs
exist in other plant species KNAT1 is expressed
naturally only in the shoot meristem Constitutiv
e (35S-driven) ectopic expression resulted in
shoot meristem initiation in leaves Chuck et al.
(1996) Plant Cell 81277-1289 (examples)
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35SKNAT1 Expression Mediates Adventitious Shoot
Formation in Arabidopsis

• Transverse section of a wild-type rosette leaf,
  including the midvein. Bar 100 ?m.
• (B) Transverse action of a 35SKNAT1 rosette
  leaf, including the midvein and enlarged
  secondary veins. Bar 100 ?m.
• Close-up of a transverse section from the wild
  type showing palisade and spongy parenchyma. Bar
  50 ?m.
• Close-up of a transverse section from a
  35SKNAT1 leaf showing an abnormal vein, tightly
  packed cells, and lack of palisade parenchyma.
  Bar 50 ?m.
• Transverse section of a cauline leaf from a
  35SKNAT1 transformant with an initiating
  inflorescence meristem over a vein. Bar 25 ?m.
• Transverse section of a 35SKNAT1 rosette leaf
  with an initiating vegetative meristem over a
  vein. A procambial strand is differentiating
  between the leaf primordia and the existing vein.
  Bar 25 ?m.

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KNAT1 Expression Is Correlated with Development
of an Adventitious Shoot Meristem (Fig. 6A and B)
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• Genes that regulate plant regeneration
  identification of genes that regulate plant
  development may result in new approaches for
  plant regeneration in vitro
• Adventitious Shoot Formation
• KNAT1 family transcription factors
• Cytokinin signaling and shoot effector genes,
  etc., illustration
• Somatic Embryogenesis
• SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)
• Transcription factors WUSCHEL, BABYBOOM, LEAFY
  COTYLEDON 1/2, DORNROSCHEN/ENHANCER OF SHOOT
  REGERATION1

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Positive and Negative Regulators of Shoot
Formation in Arabidopsis
(Cytokinin Overproducing)
Howell et al. (2003) Trends Plant Sci 8453-459
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• Genes that regulate plant regeneration
  identification of genes that regulate plant
  development may result in new approaches for
  plant regeneration in vitro
• Adventitious Shoot Formation
• KNAT1 family transcription factors
• Cytokinin singaling and shoot effector genes,
  etc.
• Somatic Embryogenesis
• SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)
• Transcription factors WUSCHEL, BABYBOOM, LEAFY
  COTYLEDON 1/2, DORNROSCHEN/ENHANCER OF SHOOT
  REGERATION1

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II. Culture medium Basal medium - essential
micro- and macronutrients, carbon source
(sucrose/glucose), thiamine-HCl, and
i-inositol NH4 may facilitate organogenesis,
NO3- favors embryogenesis, and glutamine/asparagin
e/proline favor embryogenesis. Growth
regulators - auxin and cytokinin are the critical
components for regulation of morphogenesis in
vitro Hormonal requirements differ depending on
the morphogenetic process Organogenesis En
hancement of axillary bud proliferation -
(Wickson and Thimann, 1958) - cytokinin
antagonizes apical dominance caused by auxin,
example
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Adventitious shoot/root formation (Skoog and
Miller, 1957) High cytokinin/low auxin shoot
formation/caulogenesis Low cytokinin/high auxin
root formation/rhizogenesis Meristemoids are
competent by not committed to development,
examples
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Auxin and Cytokinin Regulation of Organogenesis,
Tomato Leaf Discs
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Auxin and Cytokinin Regulation of Organogenesis
(Gloxinia Leaf Segments)
BA 0.3 0.3 0.3
0.3 0.3 (mg/L)
NAA 0 0.1 0.3
1.0 3.0
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Somatic embryogenesis (Reinert, and Steward et
al., 1958) Carrot callus, 2,4-D either favors
proliferation of competent cells or induces
acquisition of competence by cells, example
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Embryogenesis/Embryogeny of Carrot Is Regulated
by 2,4-D (mg/L)
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Gibberellin - inhibits differentiation of
meristmoids but may be required for shoot
development, example
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Stage II
Stage I
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• III. Culture Environment
• Light
• Intensity - darkness or low light (20 ?mol m-2
  s-1 or 1000 lux)
• Photoperiod - 16 hours daily
• Quality - blue wavelength favors shoot
  organogenesis, red wavelength favors root
  organogenesis
• Temperature
• Absolute - 24-27C is standard, however, cool
  weather species may respond better to lower
  temperatures, e.g. potato - 18C for shoot
  induction and 27C for development
• Diurnal fluctuation - not usually important
• Dormancy treatment can be in culture, e.g.
  bulbs and vernalization of scales, example

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Cold Treatment of In Vitro Cultured Lily Bulb
Scale Explants Satisfies Dormancy Requirement
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4. Genetic Variation in Regenerated Plant
Populations

• Background - Phenotypic variation in regenerated
  plant populations can be epigenetic
  (non-genetic/unstable) or genetic (inherited
  through gametes)
• Genetic variation in regenerated plants can arise
  because cells in the explant are genetically
  variant or tissue culture has proliferated
  variant cells or induced variation, example

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4. Genetic Variation in Regenerated Plant
Populations

• Regulating genetic variation in regenerated plant
  populations
• Low variability - highly meristematic explants
• Selection and reculture of meristmatic propagules
• Rapid regeneration without sustained periods in
  culture
• Simple media with low concentrations of growth
  regulators
• High variability - explants containing
  differentiated cells
• Long passage intervals where cells go through
  division and expansion phases
• Sub-culturing non competent cells, high
  concentrations of growth regulators, example

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Regulating Genetic Variability in Regenerated
Plants
w/selection
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Chimerism - plants exhibiting sectorial or
periclinal chimeras can result from adventitious
shoot initiation since morphogenesis may be
initiated from more than one cell, example
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5. Regeneration of Cereals

• Background - morphogenesis is focused on
  producing transgenic plants
• Isolation, culture and maintenance of competent
  cells and regeneration of transgenic plants
• Embryogenesis is preferred because of single
  cell origin.
• Phase/stages of culture leading to plant
  regeneration
• Induction
• Maintenance
• Regeneration
• Rooting

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5. Regeneration of Cereals
Phases/stages of culture leading to plant
regeneration Induction Maintenance Regen
eration Rooting
Induction - explants are isolated that contain
high frequency of competent cells and there is
proliferation of pre-embryonically competent
cells (PEDC) Medium with high auxin and, in some
instances, asparagine/ proline/glutamine, examples
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Embryogenic Competence of Sorghum Immature
Inflorescences
Notes data obtained from experiments conducted
in April 1995, with sorghum genotype PHB82.
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Embryogenic Competence of Sorghum Immature Embryos
Notes data obtained from experiments conducted
in April 1996, with sorghum genotype PH391.
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• Maintenance - competent cells continue to
  proliferate and differentiation occurs
• Cells tends to become non competent with time
• Visual selection pressure is applied
• Medium favors embryogeny and shoot formation
  (lower auxin cytokinin), example
• Regeneration - plant development, lower cytokinin
  auxin
• Rooting - root development in somatic embryos,
  minimal or no cytokinin and w/o or w/auxin

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Induction and Maintenance of Embryogenic Callus
from Sorghum Immature Inflorescences or Embryos
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• Regeneration - plant development, lower cytokinin
  auxin
• Rooting - root development in somatic embryos,
  minimal or no cytokinin and w/o or w/auxin,
  example

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Regeneration of Sorghum via Somatic Embryogenesis
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Cereal Culture Plant Regeneration Stages, Sorghum
Example
w/selection for morphogenic callus
w/selection