Plant Cell, Tissue and Organ Culture

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Plant Cell, Tissue and Organ Culture Hort
515 Embryo, Meristem, and Root Cultures

• Embryo Culture culture of zygotic embryos to
  recover plants, i.e. germination of embryos that
  are dormant or must be rescued at very immature
  stages of development (hybrids of wide crosses)
• Meristem Culture excision and culture of the
  shoot apical meristem to recover disease-free
  plants
• 3. Root Culture autonomously growing roots for
  production of secondary products

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• Embryo Culture

I. Germination of dormant embryos - typically
the result of either chemicals produced in the
ovary/ovule, physical/chemical barriers to seed
germination or dormancy programs Seed dormancy
requirement may be satisfied by hormone or
stratification treatments in vitro Orchid
(epiphyte) seeds do not have an endosperm but
nutrients can be supplied in a tissue culture
medium (e.g. banana pulp). II. Rescue of
immature embryos - these are products of wide
crosses that are exhibiting some incompatibility
responses that prevent development of a mature
embryo, i.e. products of parents in secondary
gene pools, example
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Pre-fertilized Ovule
Two male gametes, one fertilizes the egg to make
a zygote and the other fuses with the polar
nuclei forming the triploid endosperm.
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II. Rescue of immature embryos
Embryo abortion based on zygotic incompatibility
barriers Gene pool classification by Harlan
and deWit Primary - no genetic barriers to
recombination Secondary - pre- and
post-zygotic incompatibility barriers,
example Tertiary - chromosomal barriers that
restrict homeologous chromosome pairing and
recombination
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II. Rescue of immature embryos
Embryo abortion based on zygotic incompatibility
barriers Gene pool classification by Harlan
and deWit Primary - no genetic barriers to
recombination Secondary - pre- and
post-zygotic incompatibility barriers,
Tertiary - chromosomal barriers that
restrict homeologous chromosome pairing and
recombination
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II. Rescue of immature embryos
Rescue of immature embryos that are products of
wide crosses is possible if the genotypes are
members of the secondary gene pool, i.e. pre- and
post-zygotic incompatibility barriers Test
tube fertilization may result in completion of
germination if there are pre-zygotic barriers
such as stylar and pollen tube length disparities
Embryo culture embryo development,
germination and seedling development if there are
post-zygotic barriers, example
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4, 5, 6 - may be overcome by test tube
fertilization 7, 8, 9 - may be rescued by embryo
culture
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Embryogenesis - embryo initiation from the
zygote first divisions are horizontal
(periclinal), separating the suspensor from the
embryo proper and then transverse (anticlinal)
divisions begin the process of differentiation,
suspensor, proembryo Embryogeny - embryo
development after differentiation,
examples Embryo abortion in wide crosses often
occurs during embryogeny (e.g. endosperm
degradation) and it is sometimes possible to
rescue these embryos and culture in vitro to
recover plants
Embryo culture may include the culture of embryos
within the ovule or ovary in which instances
test-tube fertilization may overcome stigmata or
style, and pollen incompatibility barriers
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Embryogenesis
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Embryogeny
Embryogenesis
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Embryogenesis - embryo initiation from the
zygote first divisions are horizontal,
separating the suspensor from the embryo proper
and then transverse divisions begin the process
of differentiation, suspensor, proembryo
Embryogeny - embryo development after
differentiation Embryo abortion in wide crosses
often occurs during embryogeny (e.g. endosperm
degradation) and it is sometimes possible to
culture these embryo and recover hybrid plants
Embryo culture may include the culture of embryos
within an ovule or ovary in which instances
test-tube fertilization may overcome stigmatal or
stylar, and pollen incompatibility barriers,
examples
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Tomato ovary culture
CA poppy ovule culture
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• Isolation and culture of immature embryos
• History - Hannig (1904), 1st embyro culture,
  Raphanus and Cochlearia on medium containing
  salts sucrose

Retention of the ovary on the parent plant
Embryos become more become more autotrophic
during development Plant treatments that
facilitate parthenocarpy enhance embryo
development, typically facilitated by hormones,
example
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• Isolation and culture of immature embryos

• Nutrient Medium
• Mineral nutrients essential micro- and
  micro-nutrients
• Carbohydrates - (carbon source)/osmotic agents,
  50 g/L equivalent of sucrose (normal is 20 to 30
  g/L), high osmolarity favors embryogeny and
  prevents premature germination
• Growth regulators - Auxin, cytokinin and
  gibberellins tend to be required for
  preheart-shape stage embryos
• ABA is used to prevent precocious germination,
  examples

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Embryo Culture of Japanese Holly
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Embryo Culture of Citrus
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2. Meristem Culture for Disease Eradication
Clonal propagation of plants using explants that
are free of disease organisms Typically, the
explant is the shoot apex, containing the apical
meristem, as this explant often does not contain
microbes or viruses and will regenerate shoots
potatoes, strawberries, most tuber crops, citrus
I. Background Shoot Apical Meristem - apical
portion of the shoot that contains the
progenitors of vegetative cells and subsequently
germ cells Tunica - peripheral 1 to 3 layers of
cells characterized by anticlinal divisions,
gives rise to the epidermis/subepidermis Corpus
- cells subjacent to the tunica, periclinal and
anticlinal divisions and gives rise to the
cortex, vascular system and pith Meristem
initials - 3 to 5 cells that are progenitors of
the tunica/corpus, relatively low cell division
frequency
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Dicot Shoot Apical Meristem
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Shoot apex - meristem with leaf initials, most
typically is the explant that is cultured for
disease eradication, larger in size and more
autotrophic than the true apical meristem,
example
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Shoot apical meristem is often free of viruses
and other pathogens Vasculature is not directly
connected to the meristem
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II. Factors affecting recovery of disease-free
plants
Treatment of the donor plant - treatments that
favor differential growth of the plant over the
disease organism

• Gibberellin or etiolation treatments facilitate
  more rapid growth of the shoot
• Thermotherapy treatment of plants reduces
  pathogen growth (viral replication), 35 to 42 C
  constant or fluctuating for 3 to 6 weeks, example

Nutrient medium - Assuming that a shoot apex is
cultured, then basal medium a low level of
cytokinin to promote shoot elongation and
axillary bud development, gibberellin may also
favor shoot elongation
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Thermotherapy and Tissue Culture Procedures for
Obtaining Disease-free Stock Plants
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II. Factors affecting recovery of disease-free
plants
Treatment of the donor plant - treatments that
favor differential growth of the plant over the
disease organism

• Gibberellin or etiolation treatments facilitate
  more rapid growth of the shoot
• Thermotherapy treatment of plants reduces
  pathogen growth (viral replication), 35 to 42 C
  constant or fluctuating for 3 to 6 weeks

Nutrient medium shoot apex culture basal medium
a low level of cytokinin to promote shoot
elongation and axillary bud development,
gibberellin may also favor shoot elongation,
example shoot apical meristems require more
complex media
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Asparagus Shoot Apex Development Stimulated by
Low Cytokinin Auxin
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3. Root Cultures

• Definition and Background
• Explant, Media, Growth Conditions, and Reculture
• III. Hairy Root Cultures

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3. Root Cultures
I. Definition and Background
Roots growing autonomously in vitro P R White
established the first root culture (tomato) in
1933, culture is still maintained (1980), even
though the primary root meristem has a
determinate growth pattern Principal use was
to study the physiology and metabolism of roots,
and primary root determinate growth
patterns Transformation to produce hairy root
cultures has refocused interest on root secondary
product biosynthesis
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II. Explant, Media, Growth Conditions, and
Reculture

• Explant primary root of aseptic seedling,
  example
• Media basal (essential micro- and
  macronutrients, carbon source), thiamine,
  typically growth regulator autotrophic
• Growth Conditions liquid or semisolid medium,
  aeration is important
• Reculture terminal meristem has a finite
  (determinant) growth, culture is maintained by
  re-culturing lateral root segments

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Root Culture Initiation
Seedling after germination in vitro, primary root
without secondary roots
Excise the terminal 10 mm and culture into medium
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II. Explant, Media, Growth Conditions, and
Reculture

• Explant primary root of aseptic seedling
• Media basal (essential micro- and
  macronutrients, carbon source), thiamine,
  typically growth regulator autotrophic
• Growth Conditions liquid or semisolid medium,
  aeration is important
• Reculture terminal meristem has a finite
  (determinant) growth, culture is maintained by
  re-culturing lateral root segments, example

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Root Culture Growth and Reculture
Tomato root cultures
Reculture by excising lateral root and inoculate
into fresh medium
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Reculture of a Root
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III. Hairy Root Cultures
Hairy root cultures are capable of complete
autonomous growth/proliferation because of
Agrobacterium rhizogenes transformation including
production of numerous lateral roots, example
Hairy root culture scale-up
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Hairy Root Culture
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III. Hairy Root Cultures
Hairy root cultures are capable of complete
autonomous growth/proliferation because of
Agrobacterium rhizogenes transformation including
production of numerous lateral roots Hairy
root culture scale-up - The vigorous growth of
these cultures has made scale-up by engineers
feasible Illustrated is the growth of hairy root
culture, culture vessels for scale-up and types
of products that have been produced by hairy root
cultures, examples
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Hairy Root Culture Fermentation Systems
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In this case, fast growing root cultures were
established in medium devoid of phytohormones
without being transformed with A. rhizogenes.
This serves to remind us that it is the fact that
fully differentiated roots are being cultured,
and not transformation by Ri T-DNA per se, which
accounts for the large number of reports of
secondary metabolite formation by hairy roots as
indicated in Table 1.1.