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

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

5. Morel
1960
Disease eradication
Wimber
1963
& in vitro production of orchids

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

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. Through protocorms, 1,000,000 per year.
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. Basal ‘hormone free’ medium
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

51. STAGE IV: Transfer to Natural Environment
Acclimatization:
Process whereby plants physiologically and
anatomically adjust from in vitro to ex vitro
cultural and environmental conditions
Two reasons micropropagated plants may be difficult to acclimatize ex vitro: Low photosynthetic competence
(heterotrophic nutrition)
Poor control of water loss