2. Somatic Embryogenesis
Parthenocarpy
Apomixis
In vitro somatic embryogenesis

4. Soybean – Wayne Parrot, UGA

5. Somatic Embryos
Bipolar
Not connected to explant or callus cells by vascular tissue
In most woody plants, tissue must be juvenile or reproductive

6. Indirect Somatic Embryogenesis

7. Induction
Auxins required for induction
Proembryogenic masses form
2,4-D most used
NAA, dicamba also used

8. Development Auxin must be removed for embryo development
Continued use of auxin inhibits embryogenesis
Stages are similar to those of zygotic embryogenesis
Globular
Heart
Torpedo
Cotyledonary
Germination (conversion)

9. Maturation
Require complete maturation with apical meristem, radical, and cotyledons
Often obtain repetitive embryony
Storage protein production necessary
Often require ABA for complete maturation
ABA often required for normal embryo morphology
Fasciation
Precocious germination

10. Germination May only obtain 3-5% germination
Sucrose (10%), mannitol (4%) may be required
Drying (desiccation)
ABA levels decrease
Woody plants
Final moisture content 10-40%
Chilling
Decreases ABA levels

14. Rubber tree from somatic embryo CIRAD

21. Factors that Influence SE
Genotype
Growth regulators
Carbon source
Nitrogen

22. Maturation and Germination (Conversion)

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

24. 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

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

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

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

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

29. Conventional Propagation
Cuttings
Budding, grafting
Layering

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

31. 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

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

33. 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?

34. 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)

37. 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

38. 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

39. 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

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

41. 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

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

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

45. 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

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

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

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

50. Stage 1

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

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

57. 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

58. 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

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

60. 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

61. STAGE II: Shoot Production

62. 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

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

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

65. STAGE III: Pretransplant (rooting)

66. 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

67. Stage IV

68. 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