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