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 OvuleAntipodals

Egg

Synergids

Polar nuclei

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.

5.

7.

8.

9.

4.

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.

5.

7.

8.

9.

4.

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

150 m

Asparagus Shoot Apex

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 AnthraquinonesPolyketide 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 FlavonoidsAnthocyanin

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)

Gynostemma

Saponin Fei et al. (1993)

Hyoscyamus Tropane alkaloids Flores and Filner (1985), Parr et al. (1990), Doerk-Schmitz 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)

Leontopodium

Anthocyanins and essential oils

Hook (1994)

Linum Lignans Berlin et al. (1988)

Lippia Sesquiterpenes Sauerwein et al. (1991)

Lithospermum

Naphthoquinone (shikonin)

Shimomura et al. (1991), Sim and Chang (1993)

Lobelia Piperidine alkaloidPolyacetylenes

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)

Podophyllum

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)

Trichosanthes

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.