Plant Cell, Tissue and Organ Culture
Hort 515
Embryo, Meristem, and Root Cultures
1. 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)
2. 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
1. 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
Pre-fertilized Ovule
Antipodals
Egg
Polar
nuclei
Synergids
Two male gametes, one fertilizes the egg to make a zygote and the
other fuses with the polar nuclei forming the triploid endosperm.
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
Incompatibility Barriers
6.
7.
8.
9.
4.
5.
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
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
Incompatibility Barriers
6.
7.
8.
9.
4.
5.
4, 5, 6 - may be
overcome by test tube
fertilization
7, 8, 9 - may be
rescued by embryo
culture
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
Embryogenesis
Embryogenesis
Embryogeny
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
Tomato ovary
culture
CA poppy ovule
culture
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
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
Embryo Culture of Japanese Holly
Embryo Culture of Citrus
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
Dicot Shoot Apical Meristem
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
Asparagus Shoot Apex
150 m
Shoot apical meristem is often free of viruses and other
pathogens
Vasculature is not directly connected to the meristem
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
Thermotherapy and Tissue Culture Procedures for
Obtaining Disease-free Stock Plants
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
Asparagus Shoot Apex Development Stimulated
by Low Cytokinin + Auxin
3. Root Cultures
I.
Definition and Background
II.
Explant, Media, Growth Conditions, and Reculture
III. Hairy Root Cultures
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
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
Root Culture Initiation
Seedling after germination in vitro, primary root
without secondary roots
Excise the terminal
10 mm and culture
into medium
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
Root Culture Growth and Reculture
Tomato root cultures
Reculture by excising lateral root and inoculate into
fresh medium
Reculture of a Root
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
Hairy Root Culture
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
Hairy Root Culture Fermentation Systems
Table 1.1.
Examples of secondary metabolites produced by hairy
roots.
Genus
Metabolite
Reference
Ajuga
Hydroxyecydsone
Tanaka and Matsumoto (1993)
Ambrosia
Thiophenes
Flores et al. (1988)
Armoracia
Fusicoccin
Babakov et al. (1995)
Artemisia
Artemisinin
Qin et al. (1994), Weathers et al. (1994), Jaziri et al. (1995)
Astragalus
Astragalosides
Hirotani et al. (1994)
Atropa
Tropane alkaloids
Kamada et al. (1986), Jung and Tepfer (1987), Sharp and Doran
(1990)
Beta
Betalain pigments
Hamill et al. (1986), Taya et al. (1992, 1994)
Bidens
Polyacetylenes
Marchant (1988)
Brugmansia
Tropane alkaloid
Giulietti et al. (1993)
Campanula
Polyacetylenes
Tada et al. (1996)
Carthamus
Thiophenes
Flores et al. (1988)
Cassia
Anthraquinones
Polyketide pigments
Asamizu et al. (1988)
Ko et al. (1995)
Catharanthus
Indole alkaloids
Parr et al. (1988), Toivonen et al. (1989), Bhadra et al. (1993),
Sim et al. (1994), Jung et al. (1994)
Centranthus
Valepotriates
Gränicher et al. (1995b)
Chaenactis
Polyines
Constabel and Towers (1988)
Cinchona
Indole alkaloids
Hamill et al. (1989)
Coreopsis
Polyacetylenes
Marchant (1988)
Datura
Tropane alkaloids
Payne et al. (1987), Christen et al. (1989), Robins et al. (1990),
Parr et al. (1990), Dupraz et al. (1994), Rhodes et al. (1994)
Sesquiterpenes
Furze et al. (1991)
Daucus
Flavonoids
Anthocyanin
Bel-Rhlid et al. (1993)
Kim et al. (1994)
Digitalis
Cardioactive glycosides
Saito et al. (1990)
Duboisia
Tropane alkaloid
Deno et al. (1987b), Mano et al. (1989), Yukimune et
al. (1994)
Echinacea
Alkamides
Trypsteen et al. (1991)
Fragaria
Polyphenol
Motomori et al. (1995)
Glycyrrhiza
Glycyrrhizin
Ko et al. (1989)
Gynostemm
a
Saponin
Fei et al. (1993)
Hyoscyamus
Tropane alkaloids
Flores and Filner (1985), Parr et al. (1990), DoerkSchmitz et al. (1994)
Piperidone alkaloids
Sesquiterpenes
Sauerwein et al. (1991)
Signs and Flores (1989)
Lactuca
Sesquiterpene lactones
Kisiel et al. (1995), Song et al. (1996)
Leontopodiu
m
Anthocyanins and
essential oils
Hook (1994)
Linum
Lignans
Berlin et al. (1988)
Lippia
Sesquiterpenes
Sauerwein et al. (1991)
Lithospermu
m
Naphthoquinone
(shikonin)
Shimomura et al. (1991), Sim and Chang (1993)
Lobelia
Piperidine alkaloid
Polyacetylenes
Yonemitsu et al. (1990)
Jshimaru et al. (1994), Tada et al. (1995a), Yamanaka
et al. (1996)
Lotus
Condensed tannins
Carron et al. (1994)
Nicotiana
Pyridine alkaloids
Sesquiterpenoids
Hamill et al. (1986), Parr and Hamill (1987), Hamill et al.
(1990), Green et al. (1992), Larsen et al. (1993)
Wibberley et al. (1994)
Panax
Saponins
Yoshikawa and Furuya (1987), Inomata et al. (1993)
Platycodon
Polyacetylenes
Tada et al. (1995b)
Podophyllu
m
Lignans
Berlin et al. (1988)
Rauwolfia
Indole alkaloids
Benjamin et al, (1994)
Rubia
Anthraquinone
Sato et al (1991), van der Heijden et al. (1994), Kino-oka et al. (1994)
Rudbeckia
Thiophenes
Flores et al. (1988), Daimon and Mu (1995)
Salvia
Diterpenoid
Hu and Alfermann (1993)
Scoparia
Methoxybenzoxazolinone
Hayashi et al. (1994)
Scopolia
Tropane alkaloids
Mano et al. (1986), Parr et al (1990), Ahn et al. (1993)
Senecio*
Pyrrolizidine
Toppel et al. (1987), Hartmann and Toppel (1987)
Serratula
Ecdysteroid
Delbecque et al. (1995)
Sesamum
Naphthoquinone
Ogasawara et al. (1993)
Solanum
Steroids
Subroto and Doran (1994), Alvarez et al. (1994), Drewes and van Staden
(1995b), Ikenaga et al. (1995), Yu et al. (1996)
Swainsona
Swainsonine
Ermayanti et al. (1994)
Tagetes
Thiophenes
Westcott (1988), Croes et al. (1989), Buitelaar et al. (1993), Talou et al.
(1994), Jacobs et al. (1995)
Trichosanthe
s
Bryonolic acid
Takeda et al. (1994)
Valeriana
Valepotriates
Iridoid diester
Gränicher et al. (1994)
Gränicher et al. (1995a)
Withania
Withanolides
Banerjee et al. (1994)
*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.