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Tissue culture techniques. Embryo and Embryogenesis. Direct and indirect somatic embryogenesis. General rules for somatic embryogenesis. Factors influences somatic embryogenesis. Differences between somatic and zygotic embryogenesis. Callus and callus culture. Cell suspension culture. Somatic embryogenesis via callus and cell suspension culture. Advantages and disadvantages of somatic embryogenesis Slide 3: Plant Tissue Culture - Definition : The growth and development of plant seeds, organs, explants, tissues, cells or protoplasts on nutrient media under sterile (axenic) conditions, is known as Plant tissue culture. it is grown on a special culture medium which supports its growth and development. the medium can be either semisolid, such as agar, or liquid, such as purified water. : it is grown on a special culture medium which supports its growth and development. the medium can be either semisolid, such as agar, or liquid, such as purified water. Tissue culture produces clones, in which all product cells have the same genotype (unless affected by mutation during culture). Characteristics of Plant Tissue Culture Techniques: : Characteristics of Plant Tissue Culture Techniques: Environmental condition optimized (nutrition, light, temperature). Ability to give rise to callus, embryos, adventitious roots and shoots. Ability to grow as single cells (protoplasts, microspores, etc.). Plant cells are totipotent, able to regenerate a whole plant. Why Plant Tissue Culture is Important? : Why Plant Tissue Culture is Important? It has made significant contributions in: The production of plant materials. Plant breeding Gene banks The production of chemical compounds (secondary metabolites) etc. Tissue culture clones are ‘true to type’ as compared with seedlings, which show greater variability. Tissue culture allows fast selection for crop improvement -explants are chosen from superior plants, then cloned. Tissue Culture Techniques: : Tissue Culture Techniques: Micro propagation . Production of pathogen free plants. Meristem Culture. Somatic embryogenesis. Organ Culture. Callus Culture. Embryo culture. Anther and pollen grain Culture Protoplast culture etc. Slide 9: Fig: Different ways of plant propagation by tissue culture Our main concern will be : : Our main concern will be : Embryogenesis (somatic embryogenesis). Callus and cell suspension culture. Embryo : : Embryo : An embryo is made up of actively growing cells and the term is normally used to describe the early formation of tissue in the first stages of growth. Features of Embryo: : Features of Embryo: Embryo has a bipolar axis. shoot Meristem at one end of the axis. root Meristem at the other end of the axis. with the hypocotyls in the middle. has cotyledons. Rice embryo Types of embryo : : Types of embryo : Kohlenbach (1978) has proposed the following classification of embryo : Zygotic embryos – those formed by fertilized egg or the zygote. Non-zygotic embryos –those formed by cells other than the zygote. (a) somatic embryos –those formed by the sporophytic cells (except zygote) either in vitro or in vivo. (b) parthenogenetic embryos – those formed by unfertilized egg. (c) androgenic embryos – those formed by the male gametophyte (micro spore pollen grain). Embryogenesis: : Embryogenesis: Plant embryogenesis refers to the process of development of plant embryos, being either a sexual or asexual reproductive process that forms new plants. Embryogenesis may occur naturally in the plant as a result of sexual fertilization, and these embryos are called zygotic embryos and develop into seeds, which can germinate and give rise to seedlings. Plant cells can also be induced to form embryos in plant tissue culture; these embryos are called somatic embryos. Stages of embryogenesis : Stages of embryogenesis Types of embryogenesis : : Types of embryogenesis : There are two types of embryogenesis : Zygotic embryogenesis. Somatic embryogenesis. Zygotic embryogenesis : : Zygotic embryogenesis : The zygotic embryo is formed following double fertilization of the ovule, forming the plant embryo and the endosperm which together go on to develop into a seed, this process is known as zygotic embryogenesis. Seeds may also develop without fertilization through pathways referred to as apomixis Somatic embryogenesis : : Somatic embryogenesis : When embryos regenerate from somatic cells or tissues (which are haploid, diploid etc.) it is termed as Somatic Embryogenesis. Somatic embryogenesis is a process by which the somatic cells or tissues develops into differentiated embryos. Slide 19: Somatic embryogenesis was first induced in suspension culture (Stewart et al,1958) and in callus culture (Reinert,1959) of carrot. In addition to the members of Umbelliferae and Solanaceae , a range of dicotyledonous families have produced somatic embryos . SE occurs most frequently in tissue culture and as an alternative ORGANOGENESIS for regeneration of whole plant. Slide 20: In the literature ,somatic embryos are referred to by many names such as embryo like structures, adventitious or vegetative embryos , embryoids; and the process is termed as adventitious ,asexual or somatic embryogenesis. Stages of somatic embryogenesis : : Stages of somatic embryogenesis : Globular Stage: Embryo is small and round (multicellular). Heart Stage (bilateral symmetry) : shape changes to a heart shape with more cotyledon development torpedo-shaped stage – consists of initial cells for the shoot/root meristem Mature Stage: embryo becomes cylindrical Induction Development &maturation Globular Heart stage Torpedo Germination (conversion) Induction : Induction Auxin required for induction Pro-embryogenic masses are formed. 2,4-D mostly used. NAA, dicamba are also used. Development : Development Auxin must be removed for embryo development. Continuous use of Auxin inhibits embryogenesis. Stages are similar to those of zygotic embryogenesis Globular Heart Torpedo Cotyledonary Germination (conversion). Maturation : 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. Precocious germination Slide 25: Fig: The morphological stages of somatic embryo development in alfalfa (Medicago sativa L.) Routes of somatic embryogenesis : : Routes of somatic embryogenesis : Two routes to somatic embryogenesis (Sharp et al., 1980) Direct embryogenesis The embryos initiate directly from explants in the absence of callus formation. Indirect embryogenesis Callus from explants takes place from which embryos are developed. Types of embryogenic cells: : Types of embryogenic cells: Pre-embryogenic determined cells, PEDCs The cells are committed to embryonic development and need only to be released. Such cells are found in embryonic tissues. Induced embryogenic determined cells, IEDCs In majority of cases embryogenesis is through indirect method. Specific growth regulator concentrations and/or cultural conditions are required for initiation of callus and then redetermination of these cells into the embryogenic pattern of development. Direct somatic embryogenesis: : Direct somatic embryogenesis: In direct, SE, the embryoids are formed directly from a cell or small group of cells without the production of an intervening callus (common among reproductive tissues). It is rare in comparison with indirect SE. Steps of direct embryogenesis : Steps of direct embryogenesis Explants Culture of explants in the medium Embryoids Maturation Germination (Bipolar structure) Complete plant Examples of direct somatic embryogenesis: : Examples of direct somatic embryogenesis: Somatic embryogenesis has been reported from many plants such as Coffea arabica Alfalfa, Daucus carota Ranunculus scleratus, Linum usitatissimum,Brassica napus,Arachis hypogea etc. Leaves,scutellum,hypocotyl,nucellus and embryo-sac etc are used as explants. Direct embryogenesis (in cassava) Slide 31: Peanut somatic embryogenesis Indirect somatic embryogenesis : : Indirect somatic embryogenesis : In Indirect SE, callus is first produced from the explants. Embryoids can then be produced from the callus tissue or from cell suspension produced from the callus. Steps of indirect embryogenesis : Steps of indirect embryogenesis Explants Culture of explants in the medium Callus formation Embryo Maturation Germination (Bipolar structure) Complete plant Examples of indirect somatic embryogenesis: : Examples of indirect somatic embryogenesis: Secondary phloem of carrot, leaf tissues of coffee, Petunia, Asparagus etc. In majority of cases embryogenesis is through indirect method. Indirect embryogenesis (in coffee) Slide 35: Fig: Steps involved in Indirect S.E. Fig: diagrammatic representation of in vitro embryogenesis in suspention cultures of wild carrot. : Fig: diagrammatic representation of in vitro embryogenesis in suspention cultures of wild carrot. Auxin removal PEM Primordium Enlarging cells Slide 37: Callus proliferation in ginger. a – white globular embryoids of the callus. b – globular and oval shaped embryos, c – club shaped embryo with cotyledon and scutellum initiation d. mature embryos showed green color e. somatic embryo germination, g – shoot and root proliferation Slide 38: Fig: schematic representation of direct and indirect somatic embryogenesis General rules for the induction of somatic embryo : : General rules for the induction of somatic embryo : A high auxin concentration is often required for embryo induction. For further development of the embryos,auxin conc. should be lowered or completely eliminated from the medium. Gibberellins and ethylene usually inhibit embryogenesis. Callus from juvenile plant are more efficient for embryogenesis. Slide 40: Reduced nitrogen in the form of ammonium ions can be an important factor in embryogenesis. Light generally promotes embryogenesis. High temperature usually favorable for somatic embryogenesis. Coconut milk often promotes embryogenesis. Abscisic acid, exerts a number of striking effects on the somatic embryos on suspension culture. Slide 41: The physiological state of the plant from which the explants is taken is extremely important, as is the season during which the material is removed. FACTORS ASSOCIATED IN S.E: : FACTORS ASSOCIATED IN S.E: Media Components. Half normal concentration of media components and in some cases lacking phytohormones. Light, temperature etc. Light intensities, particular wavelengths and photoperiods, moderate and stimulate the formation and development of embryoids. Exudates Extracts (tomato, banana, yeast / coconut water) Slide 43: Other Constituents: The amount of dissolved oxygen (DO). The effect of carbon source can influence the osmotic value. Activated charcoal is reported to increase embryogenesis. As certain tissues release volatile substances which can inhibit SE in the callus. Differences between zygotic and somatic embryo : Differences between zygotic and somatic embryo Explants : Explants Pieces of organs Leaves Stems Roots Cotyledons Specific cell types Leaf tissues Pollen Endosperm Nucellus Any portion taken from a plant that will be used to initiate a culture. It can be a portion of the shoot or of the leaves or even just some cells. Callus and callus culture : : Callus and callus culture : Callus: actively dividing, non-organized tissues of undifferentiated (sometimes differentiated) cells; often developing from a point of injury (wounding). Callus is the bunch of undifferentiated meristematic tissues. Plant can even regenerate from individual cells of this callus. Slide 50: Callus -2 types Compact -cells are densely aggregated Friable -cells are only loosely associated with each other and the callus becomes soft and breaks apart easily Slide 51: Compact callus Friable callus Factors important for callus formation : Factors important for callus formation Genotype. Nutrients. Physical factors, etc. Lack of vascular elements. Auxin and sucrose. Xylogenesis. Cytokinin and gibberellins. Callus culture: : Callus culture: Callus cultures are usually initiated from tissues or organ explants after varying periods of incubation on agar culture media. At the meristematic state, a phenomenon known as de-differentiation occurs where the explants increases in size as new cells are formed by mitosis and cytokinesis. Cell division and cell expansion eventually result in the formation of callus tissue over the surface and the cut ends of the explants. cell culture : cell culture callus is placed into a liquid medium (usually the same composition as the solid medium used for the callus culture) and then agitated, single cells and/or small clumps of cells (aggregates) are released into the medium. under the correct conditions, these released cells continue to grow and divide, eventually producing a cell-suspension culture. Cell-suspension cultures : Cell-suspension cultures Friable callus provides the inoculums to form cell-suspension cultures Cell-suspension initiation- Improved by – Manipulating the medium components Repeated sub culturing. culturing on semi-solid medium (medium with a low concentration of gelling agent) Slide 57: Friable callus Liquid medium Agitation Single cells and/or small clumps of cells are released Grow and divide, eventually Produce a cell-suspension culture. Fig: Steps involved in cell suspension culture Somatic embryos formation from cell and suspension culture : Somatic embryos formation from cell and suspension culture Culturing friable callus to a liquid medium Under agitation single cell break off and by division form cell chains and clamps which fracture again to give single cells and other small cell group. A non-organized mass of vacuolated parenchyma cells arise Transformed into cytoplasm-rich cells which become embryogenic under the influence of auxin These embryogenic tissues then transfer liquid medium. In this medium the embryogenic tissues proliferate to form somatic embryos Slide 59: Fig: protocol for somatic embryogenesis in Carrot via suspension culture Advantages of using suspension culture in SE : Advantages of using suspension culture in SE In liquid medium embryogenic tissue is covered in the media so that all tissues get more nutrients and grow rapidly. Embryos can be formed in suspension cultures in large numbers and so production can afford a very rapid means of plant propagation. Advantages of somatic embryogenesis : Advantages of somatic embryogenesis Higher propagation rate. Suitable for suspension culture. Plantlets from single genetically modified single cells. Somaclonal variation. Germplasm preservation. Labour savings. Slide 62: Synthetic seeds not commercially viable yet. • No endosperm – limited shelf life • No protective seed coat that can withstand dry conditions Artificial or synthetic seed production: Slide 63: Cultured cells Single cell origin Somatic embryo Encapsulation Somatic seeds Germination Fig: Schematic representation showing artificial or synthetic seed production Slide 64: Haploid plantlets from anther culture may obtain. Fig: Diagrammatic representation of haploid production through somatic embryogenesis Disadvantages of somatic embryogenesis : Disadvantages of somatic embryogenesis Response tissue specific (explants). Confined to few species. Inability to generate large numbers of normal, free living plantlets. Barriers to high-frequency plantlet production may occur at any of a number of points between induction and the production of a plantlets capable of surviving transfer to ex vitro condition. Slide 66: May also include, low frequency embryo production, production of malformed embryos, incomplete embryo maturation,unbreakble embryo dormancy or low plantlet vigour. May create unwanted genetic variation (Somaclonal variation). Reference: : Reference: Introduction to Plant biotechnology; H.S.Chawla Introduction to Plant Tissue Culture; Bhojwani,S.S and M.K.Razdan In vitro culture of higher plants; Pierik,R.L.M. 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