Transcript Slide 1
In Vitro Developmental Pathways Mother or donor plant drip irrigated mother plants •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 plant Explant Explant Inoculum a piece of plant material isolated from mother plant to be cultured a 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 juvenility status •influence of polarity Location Regeneration can be influenced by the position of the explant on the plant or by different physical exposure: 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 (Bonga and Durzan, and 1982) acute branch angles. 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) 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. Terminology Tissue culture is an aseptic technique asepsis avoiding contaminations using sterilization procedures axenic free from association with other living organisms sterilization killing or excluding microorganisms or their spores with heat, filters, chemicals or other sterilants 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 •HgCl2 (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 •HgCl2* 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 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°C •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. 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 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 Explants • Pieces of organs – Leaves – Stems – Roots • Specific cell types – Pollen – Endosperm – Nucellus 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 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 Callus © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS Induction © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS Division E. Sutton, UC Davis Differentiation • Organogenesis • Somatic embryogenesis Cell and Suspension Culture • Cell Cultures? • Suspension Cultures Suspension cultures • Can be initiated from any part of the plant. • Usually initiated from friable callus already growing in culture. • Transferred into liquid medium. 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 Medium • Same as for callus culture? • Gamborg B5 • Conditioning Growth Curve E. Sutton, UC Davis 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 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. Why is it possible to regenerate in vitro? • Totipotency – Initial state – Competence – Determination – Differentiation 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. Organogenesis The formation of organs (such as leaves, shoots, roots) on a plant organ, usually of a different kind. Organogenesis • Rule of thumb: Auxin/cytokinin 10:1100:1 induces roots. • 1:10-1:100 induces shoots • Intermediate ratios around 1:1 favor callus growth. Indirect organogenesis Explant → Callus → Meristemoid → Primordium 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 © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS © 1998-2003, Branch of Shemyakin&Ovchinnikov IBCh RAS Direct Organogenesis Collin & Edwards,1998, Plant Cell Culture http://www.liv.ac.uk/~sd21/tisscult/publications .htm Somatic Embryogenesis Parthenocarpy Apomixis In vitro somatic embryogenesis Soybean – Wayne Parrot, UGA Somatic Embryos • Bipolar • Not connected to explant or callus cells by vascular tissue • In most woody plants, tissue must be juvenile or reproductive Indirect Somatic Embryogenesis Induction • Auxins required for induction –Proembryogenic masses form –2,4-D most used –NAA, dicamba also used 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) 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 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 Rubber tree from somatic embryo CIRAD Factors that Influence SE • • • • Genotype Growth regulators Carbon source Nitrogen Maturation and Germination (Conversion)