34
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 Types

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

Embed Size (px)

Citation preview

Page 1: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 2: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Types of culture

Plant tissue culture

Embryo culture Seed culture Meristem culture

Protoplast cultureCell culture

Callus culture Bud culture Organ culture

Page 3: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

GreenHard

Callus Culture

Friable Callus Friable Callus

dedifferentiation redifferentiationExplant Callus Organ/embryo

Page 4: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture 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.

Page 5: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

• 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

Page 6: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 7: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

• 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

Page 8: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Sieve (300 to 500 m) to filter suspension

2nd Passage1st PassageFriable Callus

Initiation of a Cell Suspension Culture from Callus

Page 9: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Suspension Cell Culture : Suspension Cell Culture :

Embryogenic cells

Cell culture

Page 10: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Embryo culture

• Mature embryo culture–Seed dormancy (ripe seeds)

• Immature embryo / embryo rescue–To avoid embryo abortion (Hybrid embryo)

Page 11: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture 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

Page 12: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Embryo Culture of Citrus

Page 13: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Root Culture

Callus arising from root tissue

Page 14: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Apical meristem CultureFor Propagation and Virus Elimination

Page 15: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Shoot Tip Propagation of Asparagus by Enhancement of Axillary Bud Development

Page 16: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

1964 Guha & Maheshwari Anther culture ---> haploid plant

  ( Datura )  

1968 Niizeki & Oono : (Japan)

Haploid plant of rice

* Started for plant breeding

Anther culture

Page 17: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Anther and microspore culture

Page 18: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 19: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Microspore culture

• Culturing methods– pollen (microspore) culture – advantages

• less competition among microspores• no diploid anther walls• greater potential haploid plant production

Page 20: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 21: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 22: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

• 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

Page 23: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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)

Page 24: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 25: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Protoplasts Fusion

Protoplast Transformation

wall synthesis

Single cell systems

Page 26: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 27: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Protoplast Culture

Hanging-drop cultures Microculture chambers

Page 28: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

LEAF-DERIVED CITRUS PROTOPLASTS

Page 29: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 30: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 31: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 32: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

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

Page 33: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Induction and Maintenance of Embryogenic Callus from Sorghum Immature Inflorescences

Page 34: Callus Culture Cell-suspension Culture Tissue or Organ Culture Shoot tip/ apical meristem culture Axillary Bud culture Root culture Ovary culture Embryo

Regeneration of Sorghum via Somatic Embryogenesis