Culture Types Cell-suspension Culture Tissue or Organ Culture Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo culture Anther/mocrospore culture Protoplast Culture

Types of culture Embryo culture Seed culture Meristem culture Cell culture Plant tissue culture Protoplast culture Organ culture Bud culture Callus culture

Explant Callus Organ/embryo Callus Culture dedifferentiation redifferentiation Explant Callus Organ/embryo Friable Callus Friable Callus Hard Green

Callus An amorphous mass of loosely arranged thin-walled parenchyma cells arising from the proliferating cells of the parent tissue cultured on agar medium.

Cellular totipotency Cytodifferentiation Dedifferentiation Cell differentiation, mainly emphasis on vascular differentiation, tracheary element differentiation, etc. Dedifferentiation The phenomenon of mature cells reverting to a meristematic state and forming undifferentiated callus tissue. Redifferentiation The ability of the component cells of the callus to differentiate into a whole plant or organ.

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, example II. Indirect - callus is proliferated from the primary explant, dedifferentiate into meristemoids and then differentiate into shoots C. Adventitious root formation - roots are initiated adventitiously at the base of the shoot apex and a vascular continuum is established to complete plant regeneration, example II. Somatic embryogenesis

Suspension Cell Culture : A type of culture in which cells and/or clumps of cells grow and multiply while suspended in a liquid medium Rapidly dividing Homogenous cells or cell aggregates Suspended in a liquid medium Cultured to produce a “cell line”。

Initiation of a Cell Suspension Culture from Callus  Sieve (300 to 500 m) to filter suspension   Friable Callus 1st Passage 2nd Passage

Suspension Cell Culture : Embryogenic cells

Embryo culture Mature embryo culture Immature embryo / embryo rescue Seed dormancy (ripe seeds) Immature embryo / embryo rescue To avoid embryo abortion (Hybrid embryo)

Application of embryo culture Prevention of embryo abortion in wide crosses. Production of haploids Overcoming seed dormancy Shortening of breeding cycle In vitro clonal propagation

Embryo Culture of Citrus

Root Culture Callus arising from root tissue

For Propagation and Virus Elimination Apical meristem Culture For Propagation and Virus Elimination

Shoot Tip Propagation of Asparagus by Enhancement of Axillary Bud Development

Anther culture Guha & Maheshwari Niizeki & Oono : (Japan) Anther culture ---> haploid plant   ( Datura )   Niizeki & Oono : (Japan) Haploid plant of rice ＊ Started for plant breeding

Anther and microspore culture

Anther culture Culturing methods anther culture – easiest and simplest protocol for tobacco anther culture (aseptically) detach anther from tobacco filament float anther on a liquid (MS-type) culture medium

Microspore culture Culturing methods pollen (microspore) culture – advantages less competition among microspores no diploid anther walls greater potential haploid plant production

Microspore culture Culturing methods Pollen (microspore) culture – advantages less competition among microspores no diploid anther walls greater potential haploid plant production Culturing methods squeeze out microspores into liquid medium filter through nylon screen of approp. pore size (e.g., 40 μm for Brassicas) centrifuge at 50-100g for ca. 5 min. resuspend and load onto a 24%/32%/40% Percoll gradient solution and spin plate suspensions as a thin layer in petri dishes and incubate at 32° C in the dark 3-5 days, then at 25° C

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

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.

Protoplast isolation: Enzymatic method Enzyme solution : 1% Cellulase, 0.25-1% Macerozyme 27.2 mg/l KH2PO4 101 mg/l KNO3 1480 mg/l CaCl2.2H2O 246 mg/l MgSO4.7H2O 0.5M Mannitol (pH 5.6)

Protoplast isolation: Enzymatic method obtain sterile plant material rinsing in a suitable osmoticum facilitating enzyme penetration purification of the isolated protoplasts (removal of enzymes and cellular debris) transfer to a suitable medium

Protoplasts Fusion wall synthesis Protoplast Transformation Single cell systems

Protoplast Culture Protoplasts can been cultured in several ways: Hanging-drop cultures Microculture chambers Soft agar (0.75 % w/v) matrix. This is one of the better methods as it ensures support for the protoplast.

Protoplast Culture Hanging-drop cultures Microculture chambers

LEAF-DERIVED CITRUS PROTOPLASTS

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. Induction B. Maintenance C. Regeneration D. Rooting

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. Induction B. Maintenance C. Regeneration D. Rooting A. 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

Embryogenic Competence of Sorghum Immature Embryos < 1.0 398 29 Embryo Size (mm) Numbers of Explants Embryogenic Callus (% of Explants) < 1.0 398 29 1.0 - 1.4 339 45 1.5 - 2.0 141 28 > 2.0 168 15

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), example Regeneration - plant development, lower cytokinin + auxin D. Rooting - root development in somatic embryos, minimal or no cytokinin and w/o or w/auxin

Induction and Maintenance of Embryogenic Callus from Sorghum Immature Inflorescences

Regeneration of Sorghum via Somatic Embryogenesis