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2 Somatic EmbryogenesisParthenocarpy Apomixis In vitro somatic embryogenesis

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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 developmentContinued 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% germinationSucrose (10%), mannitol (4%) may be required Drying (desiccation) ABA levels decrease Woody plants Final moisture content 10-40% Chilling Decreases ABA levels

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14 Rubber tree from somatic embryo CIRAD

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21 Factors that Influence SEGenotype 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 1950sMorel & Martin 1952 Meristem-tip culture for disease elimination

26 Morel 1960 Disease eradication Wimber 1963 & in vitro production of orchids

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 PropagationCuttings Budding, grafting Layering

30 Advantages Conventional Propagation Equipment costs minimalLittle 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 rapidlyMultiplication in controlled lab conditions Continuous propagation year round Potential for disease-free propagules Inexpensive per plant once established

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

33 Micropropagation DisadvantagesSpecialized 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 varietiesElimination 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)

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

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

41 Shoot Culture Method OverviewClonal 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

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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 Through protocorms, 1,000,000 per year.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 productionSelection of plant material Establish aseptic culture Multiplication Shoot elongation Root induction / formation Acclimatization

47 Selection of plant materialPart of plant Genotype Physiological condition Season Position on plant Size of explant

48 Physiological state - of stock plantVegetative / Floral Juvenile / Mature Dormant / Active Carbohydrates Nutrients Hormones

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50 Stage 1

51 Disinfestation Stock plant preparation Washing in waterDisinfecting solution Internal contaminants Screening

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56 Mother Block: A slowly multiplying indexed and stabilized set of cultures Serve as source of cultures (explants) for Stage II multiplication

57 Stage I - SterilizationPre-treatments Transfer plants to a greenhouse to reduce endemic contaminants Force outgrowth of axillary buds Washing removes endemic surface contaminants Antibiotics, fungicides, Admire, others Bacteria and fungi will overgrow the explant on the medium unless they are removed Pre-treatments to clean up the explant Detergents Sterilants and Antibiotics

58 STAGE II: Shoot Production• Stage II selection of cytokinin type and concentration determined by: Shoot multiplication rate Length of shoot produced Frequency of genetic variability Cytokinin effects on rooting and survival

59 Problems Vitrification – a glassy appearance to tissueLong acclimation time Callus formation leading to mutations

60 STAGE II: Shoot ProductionSubculture shoot clusters at 4 -5 week intervals 3 -8 fold increase in shoot numbers Number of subcultures possible is species/cultivar dependent

61 STAGE II: Shoot Production

62 STAGE III: Pretransplant (rooting)Goals: Preparation of Stage II shoots/shoot clusters for transfer to soil (prehardening) Elongation of shoots prior to ex vitro rooting Fulfilling dormancy requirements

63 Basal ‘hormone free’ mediumShoot elongation ... Basal ‘hormone free’ medium Gibberellins Carry-over of hormones

64 Root initiation Auxins Charcoal C : N ratio Light / darknessInitiation vs growth Juvenility / rejuvenation Genotype

65 STAGE III: Pretransplant (rooting)

66 STAGE IV: Transfer to Natural EnvironmentUltimate success of shoot culture depends on ability to acclimatize vigorously growing quality plants from in vitro to ex vitro conditions

67 Stage IV

68 STAGE IV: Transfer to Natural EnvironmentAcclimatization: Process whereby plants physiologically and anatomically adjust from in vitro to ex vitro cultural and environmental conditions Two reasons micropropagated plants may be difficult to acclimatize ex vitro: Low photosynthetic competence (heterotrophic nutrition) Poor control of water loss

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