Plant tissue culture Somatic ee eeeSomatic Embryogenesis Somatic embryos are formed from plant cells that are not normally involved in the development of embryos, i.e. ordinary plant tissue. No eaboanes? Toa seed coat is formed around a somatic embryo. Applications of this process include: +clonal propagation of genetically uniform plant material + elimination of viruses sprovision of source tissue for genetic transformation; generation of whole plants from single cells called protoplasts -development of synthetic seed technology.Cells derived from competent source tissue are cultured to form an undifferentiated mass of cells called a callus. Plant growth regulators in the tissue culture medium can be manipulated to induce callus formation and subsequently changed to induce embryos to form from the callus. The ratio of different plant growth regulators required to induce callus or embryo formation varies with the type of plant. &&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.ohlenbac! as proposed the followin; classification of embryo : Zygotic embryos - those formed by fertilized egg or the zygote. “J 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).~ 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.& “*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.*Somatic embryogenesis was first induced in suspension culture (Stewart et a/,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 . ad **SE occurs most frequently in tissue culture and as an alternative ORGANOGENESIS for regeneration of whole plant.- +tnduction Globular Stage: Embryo is small and round (multicellular). Development &maturation | jeart stage (bilateral symmetry) : a Globihhr shape changes to a heart shape with more cotyledon = Heart stage development rped torpedo-shaped stage - consists =") Torpedo of initial cells for the ‘shoot/root meristem aac Mature Stage: embryo becomes cylindrical (conversion)« Auxin required for induction > Pro-embryogenic masses are formed. > 2,4-D mostly used. > NAA, dicamba are also used.ee * Auxin must be removed for embryo development. * Continuous use of Auxin inhibits embryogenesis. ~ Stages are similar to those of zygotic embryogenesis i. Globular ii. Heart iii. Torpedo iv. Cotyledonary v. Germination (conversion).* 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Fig: The morphological stages of somatic embryo development in alfalfa (Medicago sativa L.)Routes fsonticenbrygeress: ~ Two routes to somatic embryogenesis (Sharp et al., 1980) + Direct embryogenesis » The embryos initiate directly from explants in the absence of cejlus formation. % Indirect embryogenesis > Callus from explants takes place from which embryos are developed.+ 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.9 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.ieetieiceac | Explante] | Culture of explants in the medium | Embryoids Maturation | Germination (Bipolar structure) | Complete plantceertal gala ~~ « 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,nu cellus and embryo-sac etc are used as explants. 4 Direct embryogenesis (in cassavai / 1 Peanut somatic embryogenesis KiIndirect somatic embryogenesis: * In Indirect SE, catlis is first produced from the €.g, Indirect shoot formatiom from callus of tobacco explants. * Embryoids can then be produced from the callus tissue or from cell suspension produced from the callus.Steps of indirect embryogenesis. Explants , 4 Culture of explants in the medium | Callus formation | Embryo Maturation | Germination (Bipolar structure) Complete plant*« Secondary phloem of carrot, leaf tissues of coffee, Petunia, Asparagus etc. In majority of cases embryogenesis is through indirect method.oe Uncitterentated cats, Inairect somate embryogeness Fig. 73 Direct and indirect pathways of somatic embryogenesismage ote acento 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.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.FACTORS ASSOCIATED INE | = Q Media Components. Half normal concentration of media components and in some cases lacking phytohormones. Q Light, temperature etc. © Light intensities, particular wavelengths and photoperiods, moderate and stimulate the formation and development of embryoids. Q Exudates 4 Extracts (tomato, banana, yeast / coconut water)* Other Constituents: The amount of dissolved oxygen (DO). The effect of carbon sourcé ban influence the osmotic value. Activated charcoal is reported to increase embryogenesis. As certain tissues release volatile substances which can inhibit SE in the callus.cape pt (ZE) Sie seed coat ‘only form embryo1, Callus ie, do-differentiated mass of cells 5. Somatic embryos do not require Gesication & dormancy. Germination ‘of embryo & regeneration of whole plant is possible throughout the yearQ In liquid medium embryogenic tissue is covered in the media so that all tissues get more nutrients and grow rapidly. Q Embryos can be formed in suspension cultures in large numbers and so production can afford a very rapid means of plant propagation.Aantagescf somatic ombnogeness o Higher propagation rate. 4 Suitable for suspension culture. 4 Plantlets from single genetically modified single cells. [ Somacional variation. & Germplasm preservation. 4 Labour savings.OArtificial or synthetic seed production: 4 Synthetic seedShot commercially viable yet. * No endosperm - limited shelf life ¢ No protective seed coat that can withstand dry conditionsCultured cells Single ce@4) origin Encapsulation Somatic seeds Germination Fig: Schematic representation showing artificial or synthetic seed productionoo Quaploid plantlets from anther culture may obtain. . S3ee. a plantlet [on ch He ae scene Anthers cultured # “et nutrient medium haploid plantlet fe* Response tissue ee (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.* 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). a]References 4 Introduction to Plant biotechnology; H.S.Chawla a Introduction,tp Plant Tissue Culture; Bhojwani,S.S and M.K.Razdan 4 Jn vitro culture of higher plants; Pierik,R.L.M.