1. MICROPROPAGATION

2. Introduction In nature plants propagate either
Sexually (seeds generation) results heterogeneity Or
Asexually (vegetative multiplication) produce genetically identical plants.
Multiplication of genetically identical copies of a cultivar by asexual reproduction is called clonal propagation.
Via tissue culture called micropropagation,

4. Basic in vitro propagation ...

5. What is Micropropagation?
“… the asexual or vegetative propagation (multiplication) of plants in vitro “
Implies - regeneration
- multiplication
- uniformity ??

6. Micropropagation The art and science of plant multiplication in vitro
Usually derived from meristems (or vegetative buds) without a callus stage
Tends to reduce or eliminate somaclonal variation, resulting in true clones
Can be derived from other explant or callus (but these are often problematic)
Basic definition of micropropagation: The art & science of plant multiplication in vitro; an important aspect is that usually meristematic tissue is used as the explant, and a callus stage is avoided. If you avoid the callus stage, you can avoid the consequences often associated with callus culture: somaclonal variation, and true clones are a result; micropropagation can be done with other explants and even callus tissue, but other explants are usually more difficult to increase and as mentioned, a callus stage often has problems.

7. Methods of micropropagation
Axillary branching
Adventitious shoot formation (organogenesis)
Somatic embryogenesis
>95% of all micropropagation
Genetically stable
Simple and straightforward
Efficient but prone to genetic instability
Little used. Potentially phenomenally efficient

8. Axillary shoot proliferation
Growth of axillary buds stimulated by cytokinin treatment; shoots arise mostly from pre-existing meristems
Clonal in vitro propagation by repeated enhanced formation of axillary shoots from shoot-tips or lateral meristems cultured on media supplemented with plant growth regulators, usually cytokinins.
Shoots produced are either rooted first in vitro or rooted and acclimatized ex vitro

9. Features of Micropropagation
Clonal reproduction
Way of maintaining heterozygozity
Multiplication Stage can be recycled many times to produce an unlimited number of clones
Routinely used commercially for many ornamental species, some vegetatively propagated crops
Easy to manipulate production cycles
Not limited by field seasons/environmental influences
Disease-free plants can be produced
Has been used to eliminate viruses from donor plants

10. Micropropagation advantages
From one to many propagules rapidly
Multiplication in controlled laboratorium conditions
Continuous propagation year round
Potential for disease-free propagules
Inexpensive per plant once established
Precise crop production scheduling
Reduce stock plant space

11. Micropropagation 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

12. Micropropagation applications
Rapid increase of stock of new varieties
Elimination of diseases
Cloning of plant types not easily propagated by conventional methods (few offshoots/ sprouts/ seeds; date palms, ferns)
Propagules have enhanced growth features (multibranched character)

13. Micropropagation (contin)
Positives and negatives of micropropagation
positives
rapid multiplication rates
low space requirement
negatives
labor costs
expensive (equipment, facilities, supplies)
loss by contamination
danger of variation

14. Steps of Micropropagation
Stage 0 – Selection & preparation of the mother plant
sterilization of the plant tissue takes place
Stage I  - Initiation of culture
explant placed into growth media
Stage II - Multiplication
explant transferred to shoot media; shoots can be constantly divided
Stage III - Rooting
explant transferred to root media
Stage IV - Transfer to soil
explant returned to soil; hardened off

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

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

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

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

21. Physiological state - of stock plant
Vegetative / Floral
Juvenile / Mature
Dormant / Active
Carbohydrates
Nutrients
Hormones

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

24. Disinfestation Stock plant preparation Washing in water
Disinfecting solution
Internal contaminants
Screening

25. Organic ‘growth factors’ Growth regulators Gelling agent
The medium
Minerals
Sugar
Organic ‘growth factors’
Growth regulators
Gelling agent
Other additives

26. Physical Environment
Temperature
Moisture
Light

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

28. Lateral / Axillary buds Adventitious (de novo, re-differentiation)
Origins of new shoots ...
Terminal extension
Lateral / Axillary buds
Adventitious (de novo, re-differentiation)
Callus differentiation

29. Role of growth regulators ...
Cell division
Differentiation
Cell expansion
Apical dominance
auxins
abscisic acid
cytokinins
gibberelic acid
ethylene

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

31. Carry-over of hormones
Shoot elongation ...
Basal ‘hormone free’ medium
Gibberellins
Carry-over of hormones

32. 2. Establish aseptic culture 3. Multiplication 4. Elongation
STAGES
1. Selection of plant material
2. Establish aseptic culture
3. Multiplication
4. Elongation
5. Root induction / formation
6. Acclimatization

33. Juvenility / rejuvenation Genotype
Root initiation ...
Auxins
Co-factors
C : N ratio
Light / darkness
Initiation vs growth
Juvenility / rejuvenation
Genotype

34. 2. Establish aseptic culture 3. Multiplication 4. Elongation
STAGES
1. Selection of plant material
2. Establish aseptic culture
3. Multiplication
4. Elongation
5. Root induction / formation
6. Acclimatization

35. - survival of the new plant when removed from the in vitro environment
Acclimatization (hardening)
- survival of the new plant when removed from the in vitro environment

36. Micropropagation of almost all the fruit crops and vegetables is possible
Some examples: dwarfing sweet cherry, Shade trees, Ornamental shrubs, Roses, Clematis, Lilacs, Saskatoon berries, Nutraceutical Plants, Rhododendron, Azalea, mustard, corn, soybeans, wheat, rice, cotton, tomato, potato, citrus, turf, legumes

37. Advantages of Micropropagation
economical in time and space
greater output -can produce millions of uniformly flowering and yielding plants
African Biotechnologies - fruit crops banana and indoor pot flowers- 6 million pieces per year
disease free
elite plants with exceptional characteristics

38. Advantages Cont’d
facilitates safer movements of germplasm across nations - In vitro germplasm assures the exchange of pest and disease free material
great for
vegetatively reproduced crops
crops which produce few seeds or highly heterozygous seeds.

39. Uses of Micropropagation
Used to create transgenic, first generation plants
Used in horticulture to produce orchids, African Violets, lilies, and ferns
Used in nurseries to grow fruit trees, evergreens, roses, and shade trees

40. Benefits of Micropropagation
Many genetically identical plants can be created from one parent plant
Because plants are clones, the uniformity assures quality
Allows many plants to grow in a small place in a short time
In some species this method will produce healthier plants

41. Thank
You