Micropropagation

1. Micropropagation “… the art and science of multiplying plants in vitro.”

2. Plant Propagation by Tissue Culture • GJ DeKlerk, EF George, MA Hall (eds) • 3rd Edition, 2008 • Springer • http://www.springerlink.com/content/n5tm30/?p=2d1b86aedb5a437f987c7bd63962bbc8&pi=0

3. 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

4. Toward Commercial Micropropagation 1950s Morel & Martin 1952 Meristem-tip culture for disease elimination

6. Commercialization of Micropropagation 1970s & 1980s Murashige 1974 Broad commercial application

7. Clone Genetically identical assemblage of individuals propagated entirely by vegetative means from a single plant.

8. Conventional Propagation • Cuttings • Budding, grafting • Layering

9. Conventional Propagation Advantages • Equipment costs minimal • Little experience or technical expertise needed • Inexpensive • Specialized techniques for growth control (e.g. grafting onto dwarfing rootstocks)

10. Micropropagation 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

11. Micropropagation Advantages • Precise crop production scheduling • Reduce stock plant space • Long-term germplasm storage • Production of difficult-to-propagate species

12. 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?

13. 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, nandinas) • Propagules have enhanced growth features (multibranched character; Ficus, Syngonium)

16. Explant • Cell, tissue or organ of a plant that is used to start in vitro cultures • Many different explants can be used for micropropagation, but axillary buds and meristems are most commonly used

17. Choice of explant Desirable properties of an explant: • Easily sterilizable • Juvenile • Responsive to culture • Importance of stock plants • Shoot tips • Axillary buds • Seeds • Hypocotyl (from germinated seed) • Leaves

18. Methods of micropropagation >95% of all micropropagation Genetically stable Simple and straightforward Efficient but prone to genetic instability Little used, but potentially phenomenally efficient • Axillary branching • Adventitious shoot formation • Somatic embryogenesis

19. Axillary shoot proliferation Growth of axillary buds stimulated by cytokinin treatment; shoots arise mostly from pre-existing meristems

20. Shoot Culture Method Overview • 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

22. ADVANTAGES • Reliable rates and consistency of shoot multiplication • 3 -8 fold multiplication rate per month • Pre-existing meristems are least susceptible to • genetic changes

23. mericloning  A propagation method using shoot tips in culture to proliferate multiple buds, which can then be separated, rooted and planted out

24. First commercially used with orchids - conventional propagation rate of 1 per year. • Through protocorms, 1,000,000 per year. Corm (Swollen stem) Chop into pieces Maturation

25. Axillary shoot production • Selection of plant material • Establish aseptic culture • Multiplication • Shoot elongation • Root induction / formation • Acclimatization

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

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

29. Stage 1

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

35. Mother Block: A slowly multiplying indexed and stabilized set of cultures Serve as source of cultures (explants) for Stage II multiplication

36. Stage I - Sterilization Pre-treatments • Transfer plants to a greenhouse to reduce endemic contaminants • Force outgrowth of axillary buds • Washing removes endemic surface contaminants • Antibiotics, fungicides, Admire, others • Bacteria and fungi will overgrow the explant on the medium unless they are removed • Pre-treatments to clean up the explant • Detergents • Sterilants and Antibiotics

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

38. Physical Environment • Temperature • Moisture • Light

39. Problems • Contamination • Browning of the explant (phenolics) • Dormant buds (woody plants)

40. STAGE II: Shoot Production • • • Stage II selection of cytokinin type and • concentration determined by: • Shoot multiplication rate • Length of shoot produced • Frequency of genetic variability • Cytokinin effects on rooting and survival

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

42. Problems • Vitrification – a glassy appearance to tissue • Long acclimation time • Callus formation leading to mutations

43. STAGE II: Shoot Production • Subculture shoot clusters at 4 -5 week intervals • 3 -8 fold increase in shoot numbers • Number of subcultures possible is • species/cultivar dependent

44. STAGE II: Shoot Production

45. STAGE III: Pretransplant (rooting) • Goals: • Preparation of Stage II shoots/shoot clusters • for transfer to soil (prehardening) • Elongation of shoots prior to ex vitro rooting • Fulfilling dormancy requirements

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

47. Root initiation • Auxins • Charcoal • C : N ratio • Light / darkness • Initiation vs growth • Juvenility / rejuvenation • Genotype

48. STAGE III: Pretransplant (rooting)

49. STAGE IV: Transfer to Natural Environment Ultimate success of shoot culture depends on ability to acclimatize vigorously growing quality plants from in vitro to ex vitro conditions

50. Stage IV