MICROPROPAGATION

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)

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

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

18. Tip bud Starting material formicropropagation 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. 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. The medium • Minerals • Sugar • Organic ‘growth factors’ • Growth regulators • Gelling agent • Other additives

26. Physical Environment • Temperature • Moisture • Light

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

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

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

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

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

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

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

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

35. 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 vitrogermplasm 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