1. MICROPROPAGATION

2. Plant Tissue Culture
The culture of plant seeds, organs, tissues, cells, or protoplasts on nutrient media under sterile conditions.

3. Basis for Plant Tissue Culture
Two Hormones Affect Plant Differentiation:
Auxin: Stimulates Root Development
Cytokinin: Stimulates Shoot Development
Generally, the ratio of these two hormones can determine plant development:
 Auxin ↓Cytokinin = Root Development
 Cytokinin ↓Auxin = Shoot Development
Auxin = Cytokinin = Callus Development

4. Breeding Applications of Tissue Culture
Micropropagation
Germplasm Preservation
Somaclonal variation
Embryo Culture
Haploid Production
In vitro Hybridization- Protoplast fusion

5. Micropropagation In vitro Clonal Propagation.
Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods.

6. Clone
Clone is a plant population derived from a single individual by asexual reproduction.
Clonal Propagation is the multiplication of genetically identical individuals by asexual reproduction.

7. Features of Micropropagation
Clonal reproduction
Multiplication stage can be recycled many times to produce an unlimited number of clones
Easy to manipulate production cycles
Disease-free plants can be produced

8. Rapid clonal in vitro propagation of plants:
From cells, tissues or organs
Cultured aseptically on defined media
Contained in culture vessels
Maintained under controlled conditions of
light and temperature

9. Commercialization of Micropropagation 1970s & 1980s Murashige (1974)
Broad commercial application

10. Starting material for micropropagation
Tip bud
Leaf
Axillary bud
Internode
Root
Starting material for micropropagation

11. Selection of plant material
Part of plant
Genotype
Physiological condition
Season
Position on plant
Size of explant

12. The Medium Minerals Sugar Organic ‘growth factors’ Growth regulators
Gelling agent
Other additives

13. Physical Environment
Temperature
Moisture
Light

14. Stages 1. Selection of plant material 2. Establish aseptic culture
3. Multiplication
4. Shoot elongation
5. Root induction / formation
6. Acclimatization

15. Steps of Micropropagation
Stage I –Establishment
Selection of the explant plant
Sterilization of the plant tissue takes place
Establishment to growth medium
Stage II  - Proliferation
Transfer to proliferation media
Shoots can be constantly divided
Stage III – Rooting & Hardening
explant transferred to root media
explant returned to soil

16. Methods of Micropropagation
Organogenesis
Organogenesis via callus formation
Direct adventitious organ formation
Embryogenesis
Direct embryogenesis
Indirect embryogenesis
Microcutting
Meristem culture (Mericloning)
Bud culture

17. Organogenesis
PGRs are prob. the most important factor affecting organogenesis
cytokinins tend to stimulate formation of shoots
auxins tend to stimulate formation of roots
The central dogma of organogenesis:
a high cytokinin:auxin ratio promotes shoots and inhibits roots
a high auxin:cytokinin ratio promotes roots and/or callus formation while inhibiting shoot formation

18. Organogenesis
The process of initiation and development of a structure that shows natural organ form and function.
The ability of non-meristematic plant tissues to form various organs de novo.
The production of roots, shoots or leaves.
These organs may arise out of pre-existing meristems or out of differentiated cells.
This, like embryogenesis, may involve a callus intermediate but often occurs without callus.

19. Somatic Embryos
Tissue culture maintains the genetic of the cell or tissue used as an explant.
Tissue culture conditions can be modified to cause to somatic cells to reprogram into a bipolar structure.
These bipolar structures behave like a true embryo - called somatic embryos.

20. An Embryo is made up of actively growing cells and the term is normally used to describe the early formation of tissue in the first stages of growth.

21. Somatic Embryogenesis
The process of initiation and development of embryos or embryo-like structures from somatic cells
The production of embryos from somatic or “non-germ” cells.
Usually involves a callus intermediate stage which can result in variation among seedlings

22. Somatic embryogenesis
Somatic embryogenesis is a useful regeneration pathway for many monocots and dicots, but is especially useful for the grasses
Types of embryogenesis
zygotic embryogenesis – the result of normal pollination and fertilization
somatic embryogenesis – embryos from (cultured) sporophytic cells , that is embryos arise indirectly

23. The composition of the culture medium controls the process-
auxin (usually 2,4-D) added causes induction, the formation of embrygogenic clumps or proembryogenic masses (PEMs) (induction medium)
auxin is deleted and the clumps become mature embryos (maturation medium)

24. Stages of development
early cell division doesn't follow a fixed pattern, unlike with zygotic embryogenesis
later stages are very similar to zygotic embryos (dicot pattern)
globular stage (multicellular)
heart-shaped stage (bilateral symmetry) – bipolarity
torpedo-shaped stage – consists of initial cells for the shoot/root meristem

25. Stages of Somatic embryo development

26. Somatic Embryogenesis
Stimulation of callus or suspension cells to undergo a developmental pathway that mimics the development of the zygotic embryo.

27. Advantages From one to many propagules rapidly.
Multiplication in controlled lab conditions.
Continuous propagation year round.
Potential for disease-free propagules.
Inexpensive per plant once established.

28. Disadvantages Specialized equipment/facilities required.
More technical expertise required.
Protocols not optimized for all species.
Plants produced may not fit industry standards.
Relatively expensive to set up.

29. Micropropagation Limitations
Equipment/facility intensive operation
Technical expertise in management positions
Protocols not optimized for all species
Liners may not fit industry standard
Propagules may be too expensive

30. Applications Rapid increase of stock of new varieties.
Elimination of diseases.
Cloning of plant types not easily propagated by conventional methods.
Propagules have enhanced growth features (multibranched character;Ficus, Syngonium)