4 CallusAn amorphous mass of loosely arranged thin-walled parenchyma cells arising from the proliferating cells of the parent tissue cultured on agar medium.
5 Cellular totipotency Cytodifferentiation Dedifferentiation Cell differentiation, mainly emphasis on vascular differentiation, tracheary element differentiation, etc.DedifferentiationThe phenomenon of mature cells reverting to a meristematic state and forming undifferentiated callus tissue.RedifferentiationThe ability of the component cells of the callus to differentiate into a whole plant or organ.
6 II. Somatic embryogenesis I. Organogenesis - shoot initiation and development with subsequent formation of adventitious roots; (adventitious - initiation from cells that are not normally the progenitors)Adventitious shoot formation - dedifferentiation and/or differentiation and development of shoots from non-meristematic cells (one or more than one) either:I. Direct - cells of explant dedifferentiate (meristemoids) and then differentiate into adventitious shoots w/o callus, exampleII. Indirect - callus is proliferated from the primary explant, dedifferentiate into meristemoids and then differentiate into shootsC. Adventitious root formation - roots are initiated adventitiously at the base of the shoot apex and a vascular continuum is established to complete plant regeneration, exampleII. Somatic embryogenesis
7 Suspension Cell Culture : A type of culture in which cells and/or clumps of cells grow and multiply while suspended in a liquid mediumRapidly dividingHomogenous cells or cell aggregatesSuspended in a liquid mediumCultured to produce a “cell line”。
8 Initiation of a Cell Suspension Culture from Callus Sieve (300 to 500 m) to filter suspensionFriable Callus1st Passage2nd Passage
18 Anther culture Culturing methods anther culture – easiest and simplest protocol for tobacco anther culture(aseptically) detach anther from tobacco filamentfloat anther on a liquid (MS-type) culture medium
19 Microspore culture Culturing methods pollen (microspore) culture – advantagesless competition among microsporesno diploid anther wallsgreater potential haploid plant production
20 Microspore culture Culturing methods Pollen (microspore) culture – advantagesless competition among microsporesno diploid anther wallsgreater potential haploid plant productionCulturing methodssqueeze out microspores into liquid mediumfilter through nylon screen of approp. pore size (e.g., 40 μm for Brassicas)centrifuge at g for ca. 5 min.resuspend and load onto a 24%/32%/40% Percoll gradient solution and spinplate suspensions as a thin layer in petri dishes and incubate at 32° C in the dark 3-5 days, then at 25° C
21 Protoplast Culture: definition Isolated protoplasts have been described as "naked" cells because the cell wall has been removed by either a mechanical or an enzymatic process. In the isolated protoplast the outer plasma membrane is fully exposed
22 Protoplast isolation: Mechanical method Plasmolyzed tissues are cut &Protoplasts are released from the cut ends.Yield of viable protoplasts is meager.One advantage: deleterious effects of the wall-degrading enzymes on the metabolism of the protoplasts are eliminated.
24 Protoplast isolation: Enzymatic method obtain sterile plant materialrinsing in a suitable osmoticumfacilitating enzyme penetrationpurification of the isolated protoplasts (removal of enzymes and cellular debris)transfer to a suitable medium
25 Protoplasts Fusion wall synthesis Protoplast Transformation Single cell systems
26 Protoplast Culture Protoplasts can been cultured in several ways: Hanging-drop culturesMicroculture chambersSoft agar (0.75 % w/v) matrix. This is one of the better methods as it ensures support for the protoplast.
29 Regeneration of Cereals Background - Morphogenesis is focused primarily on producing transgenic plants. Isolation, culture and maintenance of competent cells and regeneration of transgenic plants. Embryogenesis is preferred because of single cell origin.II. Phase/stages of culture leading to plant regeneration (see example)A. InductionB. MaintenanceC. RegenerationD. Rooting
30 Regeneration of Cereals Background - Morphogenesis is focused primarily on producing transgenic plants. Isolation, culture and maintenance of competent cells and regeneration of transgenic plants. Embryogenesis is preferred because of single cell origin.II. Phase/stages of culture leading to plant regeneration (see example)A. InductionB. MaintenanceC. RegenerationD. RootingA. Induction - Explants are isolated that contain high frequency of competent cells and there is proliferation of pre-embryonically competent cells (PEDC), usually on medium with high auxin and, in some instances, asparagine/ proline/glutamine, examples
31 Embryogenic Competence of Sorghum Immature Embryos < 1.0 398 29 Embryo Size (mm)Numbers of ExplantsEmbryogenic Callus(% of Explants)< 1.0398293394514128> 2.016815
32 Maintenance - This is the period when competent cells continue to proliferate and differentiation occurs. The population of cells tends to become non competent. Selection pressure is applied. Medium favors embryogeny and shoot formation (lower auxin + cytokinin), exampleRegeneration - plant development, lower cytokinin + auxinD. Rooting - root development in somatic embryos, minimal or no cytokinin and w/o or w/auxin
33 Induction and Maintenance of Embryogenic Callus from Sorghum Immature Inflorescences
34 Regeneration of Sorghum via Somatic Embryogenesis
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