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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
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
Culture TypesCulture Types
Types of culture
Plant tissue culture
Embryo culture Seed culture Meristem culture
Protoplast cultureCell culture
Callus culture Bud culture Organ culture
GreenHard
Callus Culture
Friable Callus Friable Callus
dedifferentiation redifferentiationExplant Callus Organ/embryo
Callus
• An amorphous mass of loosely arranged thin-walled parenchyma cells arising from the proliferating cells of the parent tissue cultured on agar medium.
• Cytodifferentiation– 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.
Cellular totipotencyCellular totipotency
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
• Rapidly dividing
• Homogenous cells or cell aggregates
• Suspended in a liquid medium
• Cultured to produce a “cell line” 。
Suspension Cell Culture : Suspension Cell Culture :
A type of culture in which cells and/or clumps of cells grow and multiply while suspended in a liquid medium
Sieve (300 to 500 m) to filter suspension
2nd Passage1st PassageFriable Callus
Initiation of a Cell Suspension Culture from Callus
Suspension Cell Culture : Suspension Cell Culture :
Embryogenic cells
Cell culture
Embryo culture
• Mature embryo culture–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
Apical meristem CultureFor Propagation and Virus Elimination
Shoot Tip Propagation of Asparagus by Enhancement of Axillary Bud Development
1964 Guha & Maheshwari Anther culture ---> haploid plant
( Datura )
1968 Niizeki & Oono : (Japan)
Haploid plant of rice
* Started for plant breeding
Anther culture
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 culturePollen (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
• 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: Mechanical method
Protoplast isolation: Enzymatic method
Enzyme solution : 1. 1% Cellulase, 0.25-1% Macerozyme 2. 27.2 mg/l KH2PO4 3. 101 mg/l KNO3 4. 1480 mg/l CaCl2.2H2O 5. 246 mg/l MgSO4.7H2O 6. 0.5M Mannitol (pH 5.6)
1.obtain sterile plant material 2.rinsing in a suitable osmoticum 3.facilitating enzyme penetration 4.purification of the isolated protoplasts (removal of enzymes and cellular debris) 5.transfer to a suitable medium
Protoplast isolation: Enzymatic method
Protoplasts Fusion
Protoplast Transformation
wall synthesis
Single cell systems
Protoplasts can been cultured in several ways:
1. Hanging-drop cultures 2. Microculture chambers 3. Soft agar (0.75 % w/v) matrix.
This is one of the better methodsas it ensures support for the protoplast.
Protoplast Culture
Protoplast Culture
Hanging-drop cultures Microculture chambers
LEAF-DERIVED CITRUS PROTOPLASTS
Regeneration of Cereals
I. 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
Regeneration of Cereals
I. 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
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
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
B. 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
C. 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