Somatic Embryogenesis

2. Somatic Embryogenesis • Parthenocarpy • Apomixis • In vitro somatic embryogenesis

4. Soybean – Wayne Parrot, UGA

5. Somatic Embryos • Bipolar • Not connected to explant or callus cells by vascular tissue • In most woody plants, tissue must be juvenile or reproductive

6. Indirect Somatic Embryogenesis

7. Induction • Auxins required for induction • Proembryogenic masses form • 2,4-D most used • NAA, dicamba also used

8. Development • Auxin must be removed for embryo development • Continued use of auxin inhibits embryogenesis • Stages are similar to those of zygotic embryogenesis • Globular • Heart • Torpedo • Cotyledonary • Germination (conversion)

9. Maturation • Require complete maturation with apical meristem, radical, and cotyledons • Often obtain repetitive embryony • Storage protein production necessary • Often require ABA for complete maturation • ABA often required for normal embryo morphology • Fasciation • Precocious germination

10. Germination • May only obtain 3-5% germination • Sucrose (10%), mannitol (4%) may be required • Drying (desiccation) • ABA levels decrease • Woody plants • Final moisture content 10-40% • Chilling • Decreases ABA levels • Woody plants

14. Rubber tree from somatic embryoCIRAD

21. Factors that Influence SE • Genotype • Growth regulators • Carbon source • Nitrogen

22. Maturation and Germination(Conversion)

23. Micropropagation “… the art and science of multiplying plants in vitro.”

24. Rapid clonal in vitro propagation of plants: • from cells, tissues or organs • cultured aseptically on defined media • contained in culture vessels • maintained under controlled conditions of light and temperature

25. Toward Commercial Micropropagation 1950s Morel & Martin 1952 Meristem-tip culture for disease elimination

27. Commercialization of Micropropagation 1970s & 1980s Murashige 1974 Broad commercial application

28. Clone Genetically identical assemblage of individuals propagated entirely by vegetative means from a single plant.

29. Conventional Propagation • Cuttings • Budding, grafting • Layering

30. Conventional Propagation Advantages • Equipment costs minimal • Little experience or technical expertise needed • Inexpensive • Specialized techniques for growth control (e.g. grafting onto dwarfing rootstocks)

31. Micropropagation Advantages • From one to many propagules rapidly • Multiplication in controlled lab conditions • Continuous propagation year round • Potential for disease-free propagules • Inexpensive per plant once established

32. Micropropagation Advantages • Precise crop production scheduling • Reduce stock plant space • Long-term germplasm storage • Production of difficult-to-propagate species

33. Micropropagation Disadvantages • Specialized equipment/facilities required • More technical expertise required • Protocols not optimized for all species • Plants produced may not fit industry standards • Relatively expensive to set up?

34. Micropropagation Applications • Rapid increase of stock of new varieties • Elimination of diseases • Cloning of plant types not easily propagated by conventional methods (few offshoots/ sprouts/ seeds; date palms, ferns, nandinas) • Propagules have enhanced growth features (multibranched character; Ficus, Syngonium)

37. Explant • Cell, tissue or organ of a plant that is used to start in vitro cultures • Many different explants can be used for micropropagation, but axillary buds and meristems are most commonly used

38. Choice of explant • Desirable properties of an explant: • Easily sterilizable • Juvenile • Responsive to culture • Importance of stock plants • Shoot tips • Axillary buds • Seeds • Hypocotyl (from germinated seed) • Leaves

39. Methods of micropropagation >95% of all micropropagation Genetically stable Simple and straightforward Efficient but prone to genetic instability Little used, but potentially phenomenally efficient • Axillary branching • Adventitious shoot formation • Somatic embryogenesis

40. Axillary shoot proliferation Growth of axillary buds stimulated by cytokinin treatment; shoots arise mostly from pre-existing meristems

41. Shoot Culture Method Overview • Clonal in vitro propagation by repeated enhanced • formation of axillary shoots from shoot-tips or • lateral meristems cultured on media • supplemented with plant growth regulators, • usually cytokinins. • Shoots produced are either rooted first in vitro • or rooted and acclimatized ex vitro

43. ADVANTAGES • Reliable rates and consistency of shoot multiplication • 3 -8 fold multiplication rate per month • Pre-existing meristems are least susceptible to • genetic changes

44. mericloning  A propagation method using shoot tips in culture to proliferate multiple buds, which can then be separated, rooted and planted out

45. First commercially used with orchids - conventional propagation rate of 1 per year. • Through protocorms, 1,000,000 per year. Corm (Swollen stem) Chop into pieces Maturation

46. Axillary shoot production • Selection of plant material • Establish aseptic culture • Multiplication • Shoot elongation • Root induction / formation • Acclimatization

47. Selection of plant material • Part of plant • Genotype • Physiological condition • Season • Position on plant • Size of explant

48. Physiological state - of stock plant • Vegetative / Floral • Juvenile / Mature • Dormant / Active • Carbohydrates • Nutrients • Hormones

50. Stage 1