Somatic Embryogenesis & Somaclonal Variations Lecture Notes

SOMATIC EMBRYOGENESIS Agriculture Biotechnology (Dr. Sheeba Naz)  Somatic Cell  Somatic cell means “body” or vegetative cell, is any biological cell forming the body of a multicellular organism other than gamete, germ cell  Somatic Embryo  Bipol...

SOMATIC EMBRYOGENESIS Agriculture Biotechnology (Dr. Sheeba Naz)  Somatic Cell  Somatic cell means “body” or vegetative cell, is any biological cell forming the body of a multicellular organism other than gamete, germ cell  Somatic Embryo  Bipolar structures with both apical and basal meristematic regions, which can form shoots and roots, respectively.  Somatic Embryogenesis  Is a process by which somatic cells or tissues, including haploid cells develops into differentiated embryos and into regenerate plants.  Embryos formed by somatic embryogenesis are called embryoids.  Hybridization  Is the technique of introducing characters of two desirable species into a single offspring by means of artificial pollination.  Types of Hybridization: a) Intravarietal hybridization: Crosses between plants of the same variety. b) Intervarietal hybridization: Crosses between plants of two different varieties of the same species. c) Interspecific hybridization: Crosses between two different species of the same genus. d) Intergeneric hybridization: Crosses between the plants belongs to two different genera  Somatic Hybridization  Combining genomes of two desirable parents, irrespective of their taxonomic relationship.  A hybrid cell is formed by in-vitro protoplast fusion.  Fused protoplast developed to form a hybrid plant.  Plant protoplast has provided novel approaches, to create a new genetically modified cell.  Used in genetic manipulations or modification and improvement of the crop for somatic cells.  Can be used for different plant species  Somatic hybridization has an advantage over sexual hybridization.  Steps involved in somatic hybridization  Protoplast fusion  Hybrid cells selection  Hybrid plant identification  Applications of Somatic hybridization  Disease-resistant plants e.g. tobacco plant resistant against tobacco mosaic virus.  Environment tolerance. e.g. development of cold tolerance in tomatoes  Somatic hybrids e.g. production of high nicotine content in tobacco. Factors Affecting Somatic Embryogenesis i) Explants:  Immature zygotic embryos  Inflorescence  Cell suspension cultures  Petioles  Protoplasts  Leaves  Stems  Roots ii) Plant Growth Regulators: Auxins ( 2, 4-D the best synthetic auxin used for inducing somatic embryos) Cytokinins ( Zeatin is promotive when applied to embryogenic cells after days 3-4 transfer from the proliferation medium) iii) Nitrogen Source: Reduced form of nitrogen is the sole source of embryo formation. IV) Electrical Stimulation: Exposure of explant to mild electric current of 0.02 V DC for 20 h promoted embryogenesis V) Genotype: Genotypic variations could be due to endogenous levels of hormones  Steps  Initiation of embryogenic culture  Proliferation of embryogenic culture  Pre-maturation of somatic embryos  Maturation of somatic embryos  Plant development  Types Direct Somatic Embryogenesis:  Embryos are formed directly from explant tissue creating an identical clone without production of intervening callus.  The explants capable of direct embryogenesis seem to carry competent or “Pre-embryonic Determined Cells”(PEDCs).  These cells are committed to embryo development and need only to be released. Indirect Somatic Embryogenesis:  Explants produced undifferentiated mass of cells (callus) which is maintained or differentiated into embryo.  Specific growth regulators and culture conditions are required for callus formation and the redetermination of embryogenic development pattern called “Induced embryogenic determined cells”(IEDCs) Somatic Embryos Vs Zygotic Embryos  Somatic embryos are structurally similar to zygotic embryos found in seeds including the ability to grow into complete plants.  Somatic embryos differ in that they develop from somatic cells, instead of zygotes (i.e., fusion product of male and female gametes)  It can be used to produce duplicates of a single genotype.  Zygotic embryo, natural seed develops as a sexual process in cross-pollinating species  It is not genetically identical to one single parent.  In contrast, somatic embryo develops from somatic cells (non- sexual) and does not involve sexual recombination.  This characteristic of somatic embryos allows clonal propagation.  Specific and direct changes to be introduced into desirable elite individuals by inserting isolated gene sequences into somatic cells  Practical Applications of Somatic Embryogenesis i) Clonal Propagation ii) Raising Somaclonal Variants iii) Synthesis of Artificial Seeds iv) Source of Regenerable Protoplast System v) Genetic Transformation vi) Synthesis of Metabolites  Advantages  It is observable, as its various culture conditions can be controlled  Lack of material is not a limiting factor for experimentation  High propagation rate  Germplasm conservation  Labour saving  Elimination of diseases and viruses  SE used as a model system for the conventional plant breeding, mass propagation and the rapid genetic improvement of the commercially important crops.  Limitations  Confined to few species.  The somatic embryos show difficult germination because of their physiological and biochemical immaturity.  Instability of cultured cells in long-term cultures is a major limitation in commercial exploitation and mass propagation of SEs. Somaclonal Variations Dr. Sheeba Naz  Genetic variations in plants that have been produced by plant tissue culture and can be detected as genetic or phenotypic traits. Plants derived from such cells or progeny of such plant is called somaclones. The term somaclonal variation was first used by Larkin and Scowcroft in 1981. Phenomenon of new phenotypes occurring in plants regenerated in in-vitro culture  Basic Features of Somaclonal Variations Variations for Karyotype: (individual's collection of chromosomes) ❖ Calliclone (clones of callus) ❖ Mericlone (clones of meristem) ❖ Protoclone (clones of Protoplast) Generally heritable mutation that persist in plant population even after plantation into the field.  Mechanism of Somaclonal Variations 1. Genetic (Heritable Variations) Pre-existing variations in the somatic cells of explant Caused by mutations and other DNA changes Occur at high frequency 2. Epigenetic (Non-heritable Variations) Variations generated during tissue culture Caused by temporary phenotypic changes Occur at low frequency  Callus Tissue Organogenesis Somaclonal Variants Regenerated plants Hardening Steps involved in induction and selection of Somaclonal Variations  Causes of Somaclonal Variations Physiological Cause Genetic Cause Biochemical Cause  Physiological Cause Exposure of culture to plant growth regulators. Culture conditions  Genetic Cause 1. Change in chromosome number ▪ Euploidy: Changes/ variation in normal chromosome sets (X,2X, 3X,4X) ▪ Monoploidy: Organism with single set of chromosomes (X) ▪ Polyploidy: Organisms with more than two chromosome sets (multiple) ▪ Aneuploidy: Changes in parts of chromosome sets (one extra or missing chromosome (2n-1, 2n-2, 2n+1, 2n+2) 2. Change in chromosome structure ▪ Deletion ▪ Inversion ▪ Duplication ▪ Translocation  Autopolyploidy: possession of more than two chromosome sets derived from the same species.  Alloploidy: presence of two or more sets of chromosomes derived from two different species.  Allopolyploidy: combination of both autopolyploidy and alloploidy in which the genome may be made up of the chromosome sets of one or more species 3. Gene Mutation ▪ Tansition ▪ Transversion ▪ Insertion ▪ Deletion 4. Plasmagene Mutation Self replicating genetic material in chloroplast/ mitochondria 5. DNA Sequence Change in DNA ▪ Detection of altered fragment size by using Restriction enzyme Change in Protein ▪ Loss or gain in protein band ▪ Alteration in level of specific protein Methylation of DNA ▪ Methylation inactivates transcription process  Biochemical Cause ▪ Lack of photosynthetic ability due to alteration in: ▪ Carbon metabolism ▪ Nitrogen metabolism ▪ Antibiotic resistance  Detection and Isolation of Somaclonal Variants 1. Variant detection by cytological Studies ▪ Staining of meristematic tissues like root tip, leaf tip with acetocarmine (dying chromosomes) provide the number and morphology of chromosomes. 2. Analysis of morphological characters ▪ Qualitative characters: Plant height, maturity date, flowering date and leaf size ▪ Quantitative characters: yield of flower, seeds and wax contents in different plant parts 3. Variant detection by gel electrophoresis ▪ Change in concentration of enzymes, proteins and chemical products like pigments, alkaloids and amino acids can be detected by their electrophoretic pattern 4. Detection of disease resistance variant ▪ Pathogen or toxin responsible for disease resistance can be used as selection agent during culture. 5. Detection of herbicide resistance variant ▪ Plantlets generated by the addition of herbicide to the cell culture system can be used as herbicide resistance plant. 6. Detection of environmental stress tolerant variant ▪ Selection of high salt tolerant cell lines in tobacco ▪ Selection of water-logging and drought resistance cell lines in tomato ▪ Selection of temperature stress tolerant in cell lines in pear. ▪ Selection of mineral toxicities tolerant in plants  Advantages of Somaclonal Variations 1. Simpler, faster, and cheaper way to obtain new varieties of plants compared to classic breeding methods, it is also very useful for perennial species, with a long vegetation period 2. The variations occur with high frequency, which is advantageous compared to conventional mutagenesis. 3.Appropriate for plant species with limited genetic diversity, the generation of somaclonal variations does not require knowledge about the genome of the respective species. 4. Introduce new traits, such as resistance to a spectrum of diseases, pathotoxins, herbicides, biotic and abiotic stress. 5. Create varieties with an increased production of valuable metabolites with phototherapeutic properties. Limitations of Somaclonal Variations 1. Sometimes they are unstable and nonheritable 2. They can be associated with deleterious features, such as reduced fertility and growth rates, or low or absent powers of regeneration. 3. To confirm the stability of a cell/plant line obtained from somaclones, repeated selection is required The occurrence of Somaclonal Variation can be reduced by: Avoiding long-term cultures. Using axillary shoot induction systems where possible. Propagating chimeras by other clonal systems. Increasing numbers of subcultures increase the likelihood of somaclonal variation The number of subcultures in micropropagation protocols should be kept to a minimum. Regularly reinitiating clones from new explants, which might reduce variability over time. Avoiding 2,4-D in the culture medium, as this hormone is known to introduce variation.