1. 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

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

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

4. Callus
An 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.
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.

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, 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

7. 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”。

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

9. Suspension Cell Culture :
Embryogenic cells

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

11. 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

12. Embryo Culture of Citrus

13. Root Culture
Callus arising from root tissue

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

15. Shoot Tip Propagation of Asparagus by Enhancement
of Axillary Bud Development

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

17. Anther and microspore culture

18. 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

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

20. 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 g 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

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.

23. Protoplast isolation: Enzymatic method
Enzyme solution :
1% Cellulase, % 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)

24. 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

25. Protoplasts Fusion wall synthesis Protoplast Transformation
Single cell systems

26. 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.

27. Protoplast Culture
Hanging-drop cultures
Microculture chambers

28. LEAF-DERIVED CITRUS PROTOPLASTS

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. Induction
B. Maintenance
C. Regeneration
D. 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. 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

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

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), example
Regeneration - plant development, lower cytokinin + auxin
D. 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