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

1. 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

2. 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

3. 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.

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, example Tertiary - chromosomal barriers that restrict homeologous chromosome pairing and recombination

5. Incompatibility Barriers 6. 7. 8. 9. 4. 5.

6. 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

7. 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

8. 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

9. 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

10. Embryogenesis

12. Embryogeny Embryogenesis

13. 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

14. Tomato ovary culture CA poppy ovule culture

15. 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

17. 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

19. Embryo Culture of Japanese Holly

20. Embryo Culture of Citrus

21. 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

22. Dicot Shoot Apical Meristem

23. 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

24. Asparagus Shoot Apex 150 m

25. Shoot apical meristemis often free of viruses and other pathogens Vasculature is not directly connected to the meristem

26. 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 • Thermotherapytreatment 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

27. Thermotherapy and Tissue Culture Procedures for Obtaining Disease-free Stock Plants

28. 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 • Thermotherapytreatment 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

29. Asparagus Shoot Apex Development Stimulated by Low Cytokinin + Auxin

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

31. 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

32. 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

33. Root Culture Initiation Seedling after germination in vitro,primary root without secondary roots Excise the terminal 10 mm and culture into medium

34. 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

35. Root Culture Growth and Reculture Tomato root cultures Reculture by excising lateral root and inoculate into fresh medium

36. Reculture of a Root

37. III. Hairy Root Cultures Hairy root culturesare capable of complete autonomous growth/proliferation because of Agrobacterium rhizogenes transformation including production of numerous lateral roots, example Hairy root culture scale-up

38. Hairy Root Culture

39. 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

40. Hairy Root Culture Fermentation Systems

43. *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.