1. In Vitro Developmental Pathways

2. Mother or donor plant is a plant used as a source of explants and selected for its genotype, hygienic or physiological status see also stage O : improving hygienic and physiological status and preconditioning of mother plantstage O drip irrigated mother plants

3. Explant a piece of plant material isolated from mother plant to be cultured Inoculuma subculture of plant material which is already in culture For explants: aerial plant parts are “cleaner” than underground parts the smaller the explant the better the chances to overcome specific phytopathological problems (virus, microplasm, bacteria), but it decreases the survival rate inner tissues are less contaminated than outer ones comparable explants do not always react in a similar way influence of location on the mother plant (topophysis, cyclophysis and periphysis)influence of location on the mother plant influence of juvenility status influence of polarity

4. Location Regeneration can be influenced by the position of the explant on the plant or by different physical exposure:

5. Where are juvenile explants located? Juvenile is different from young. Young refers to the age, and is the opposite of old. Juvenile is the opposite of adult, and can refer to: the period in the lifetime that the organ was initiated (sleeping buds at the base of a stem were initiated on the seedling); the initiation period of an organ on which new structures develop (root sprouts develop on roots which were originally initiated in the seedling stage); the proximity to the root system. Juvenility in several hard-woods: Density of crosshatching indicates degree of juvenility. In the juvenile zone, note the single trunk, retention of leaves close to the trunk in winter, and obtuse branch angle. In the mature zone, note the forked trunk and acute branch angles. (Bonga and Durzan, 1982)

6. Juvenility gradients in trees Juvenility in a regulary shaped conifer: the degree of juvenility of an apical meristem is inversely proportional to the distance (along trunk and branches) between the root-shoot junction (A) and the meristem. The distance AB>AC> AD>AE>AF, and therefore, meristem B is the most mature, and meristem F is the most juvenile. (Bonga and Durzan, 1982)

7. Juvenility and propagation In vitro propagation of conifers is generally only possible with sections of embryos (A) or young germinants (B). Thus in vitro propagation capability is already lost in young seedlings (C), but these can still be propagated by conventional rooting or cutting techniques. Cuttings taken from these seedling's root readily and generally produce true-to-type propagules. Cuttings from a somewhat older tree (D) have a reduced rooting capacity, and the propagules often show varying degrees of plagiotropic growth. Cuttings from mature trees (E) do not root.

8. Terminology Tissue culture is an aseptic technique asepsisavoiding contaminations using sterilization procedures axenicfree from association with other living organisms sterilization killing or excluding microorganisms or their spores with heat, filters, chemicals or other sterilants

9. Surface sterilization of plant material An elaborated example of surface sterilization Cut the plant material to an appropriate size to fit the container which will be used during the sterilsation procedure Rinse plant material under running tap water Shake for a few seconds in alcohol HgCl 2 (0.1 - 1%) + Teepol 2 drops/100ml: 3-5 min Rinse in autoclaved distilled water Commercial bleach 7-15% + Teepol 2 drops/100ml: 10-30 min Rinse several times in autoclaved distilled water sterilization of orchid flower stalks Notes: Tap water: to remove large debris, detergent can be used to shrub the surface Ethanol: 70% is most effective to denaturate proteins. Sometimes 95% is used to dissolve the surface wax layer NaOCl (commercial bleach) or Ca(OCl) 2 : activity is based on both Cl - and other oxidation reactions HgCl 2 * activity based on Cl - and heavy metal denaturation of proteins. wetting agent (e.g. “Teepol”) can be added to the forementioned agents * Hg is toxic for the environment, therefore recuperate the Hg-solution after use and collect in a large container. Hg can be precipitated by adding ammonia to the solution, and siphoning the supernatant

10. Sterilization of culture media Thermostabile ingredients are autoclaved; thermolabile ones are filter sterilized Autoclaving: adequate autoclaving requires that the medium is maintained at a choosen temperature and at the corresponding pressure for a given time; e.g. 50 kPa corresponds to 112°C, and 100 kPa to 120°CAutoclaving Filter sterilization: disposable filter membranes are first autoclaved; the filter pore size is not greater than 0.22µm; sterilized solutions are filtered a sterile container.Filter sterilization:

11. Autoclaves Bench top model (pressure cooker type) Vertical autoclave, also used as medium preparation device Top view of left figure Vertical autocalve, laboratory model Horizontal autoclave

13. Sterilization of tissue culture containers, working tools and environment glassware: autoclaving, dry heat (180°C a few hours), radiation disposable containers: radiation paper: dry heat working tools: flaming or heat (flame, glass beads) bench surface: swapping with alcohol environment: laminar flow cabinet (HEPA-filtered) sterilizing paper: dry heat sterilizing tools laminar flow cabinet

14. Explants Pieces of organs –Leaves –Stems –Roots Specific cell types –Pollen –Endosperm –Nucellus

15. Callus Unorganized, growing mass of cells Dedifferentiation of explant –Loosely arranged thinned walled, outgrowths from explant –No predictable site of organization or differentiation Auxin + cytokinin Often can be maintained indefinitely by subculture, but may lose ability to redifferentiate Compact vs friable Habituation

16. Three stages of callus culture Induction: Cells in explant dedifferentiate and begin to divide Proliferative Stage: Rapid cell division Differentiation stage (sometimes): metabolic pathway or organogenesis

17. Callus © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS

20. Induction © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS

21. Division E. Sutton, UC Davis

22. Differentiation Organogenesis Somatic embryogenesis

23. Cell and Suspension Culture Cell Cultures? Suspension Cultures

24. Suspension cultures Can be initiated from any part of the plant. Usually initiated from friable callus already growing in culture. Transferred into liquid medium.

25. Agitation Breakdown of cell aggregates into smaller clumps of cells Maintains a uniform distribution of cells and cell clumps in the medium Provides gas exchange

26. Medium Same as for callus culture? Gamborg B5 Conditioning

27. Growth Curve E. Sutton, UC Davis

28. Batch Cultures A certain number of cells is used to inoculate the culture, in a given volume Erlenmeyer flask: volume should be about 20% of flask capacity for aeration. Roller cultures

31. Continuous Culture Bioreactors Closed continuous cultures: Remove some of the media and replace with fresh. Continuous removal or periodic. Terminate growth at harvest. Start over. Open continuous culture: Not only remove some of media, but cells too. Maintain cell density at optimal level. Can be grown for years.

32. Why is it possible to regenerate in vitro? Totipotency –Initial state –Competence –Determination –Differentiation

33. Only occurs in a few cells in culture. Why? Pre-determination prior to culture Newly formed meristems may act as sinks  Meristematic centers might actually produce compounds that inhibit neighboring cells.

34. Organogenesis The formation of organs (such as leaves, shoots, roots) on a plant organ, usually of a different kind.

36. Organogenesis Rule of thumb: Auxin/cytokinin 10:1- 100:1 induces roots. 1:10-1:100 induces shoots Intermediate ratios around 1:1 favor callus growth.

40. Indirect organogenesis Explant → Callus → Meristemoid → Primordium

41. Indirect Organogenesis Dedifferentiation –Less committed, more plastic developmental state Induction –Cells become organogenically competent and fully determined for primordia production –Change in culture conditions? Differentiation

42. © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS

47. Direct Organogenesis

48. Collin & Edwards,1998, Plant Cell Culture http://www.liv.ac.uk/~sd21/tisscult/publications.htm

49. Somatic Embryogenesis lParthenocarpy lApomixis lIn vitro somatic embryogenesis

57. Soybean – Wayne Parrot, UGA

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

59. Indirect Somatic Embryogenesis

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

61. 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)

62. Maturation Require complete maturation with apical meristem, radicle, 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

63. 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

67. Rubber tree from somatic embryo CIRAD

74. Factors that Influence SE Genotype Growth regulators Carbon source Nitrogen

75. Maturation and Germination (Conversion)