1. Explants
Sterile pieces of a whole plant from which cultures are generally initiated
Aerial plant parts are “cleaner” than underground parts
The smaller the explant the better the chances to overcome specific phytopathological problems (virus, microplasm, bacteria), but it decreases the survival rate
Inner tissues are less contaminated than outer ones
Comparable explants do not always react in a similar way , due to:
influence of location on the mother plant,
influence of juvenility status ,
influence of polarity

2. A subculture of plant material which is already in culture
Types of explant
Generally all plant cells can be used as an explant, however young and rapidly growing tissue (or tissue at an early stage of development) are preferred.
Inoculum
A subculture of plant material which is already in culture

3. Types of culture (Explant base)
Embryo culture
Seed culture
Meristem culture
Cell culture
(suspension culture)
Plant tissue culture
Protoplast culture
Organ culture
Bud culture
Callus culture

4. Types of In vitro culture (explant based)
Culture of intact plants (seed and seedling culture)
Embryo culture (immature embryo culture)
Organ culture
Callus culture
Cell suspension culture
Protoplast culture

5. Seed culture Growing seed aseptically in vitro on artificial media
Increasing efficiency of germination of seeds that are difficult to germinate in vivo
it is possible to independent on asymbiotic germination. Production of clean seedlings for explants or meristem culture

6. Embryo culture
Growing embryo aseptically in vitro on artificial nutrient media
Overcoming seed dormancy and self-sterility of seeds
Study embryo development

7. Any plant organ can serve as an explant to initiate cultures
Organ culture
Any plant organ can serve as an explant to initiate cultures
No.
Organ
Culture types 1.
Shoot
Shoot tip culture 2.
Root
Root culture 3.
Leaf
Leaf culture 4.
Flower
Anther/ovary culture

8. Shoot apical meristem culture
Production of virus free germplasm
Mass production of desirable genotypes
Facilitation of exchange between locations (production of clean material)
Cryopreservation (cold storage) or in vitro conservation of germplasm

9. Root organ culture
Production of seedling from crop which multiply through root
Production of secondary metabolite

10. Ovary or ovule culture Production of haploid plants
A common explant for the initiation of somatic embryogenic cultures
Overcoming abortion of embryos of wide hybrids at very early stages of development due to incompatibility barriers
In vitro fertilization for the production of distant hybrids avoiding style and stigmatic incompatibility that inhibits pollen germination and pollen tube growth

11. Anther and microspore culture
Production of haploid plants
Production of homozygous diploid lines through chromosome doubling, thus reducing the time required to produce inbred lines
Uncovering mutations or recessive phenotypes

12. Tissue culture is an aseptic technique
Sterilization
Killing or excluding microorganisms or their spores with heat, filters, chemicals or other sterilants
Tissue culture is an aseptic technique
Aseptic technique:
Sterile
Free of pathogenic microorganisms
Free or freed from pathogenic microorganisms
Free from the living germs of disease and fermentation
Conditions established to exclude contaminants

13. Axenic culture Germfree Uncontaminated
Free from germs or pathogenic organisms
Free from other microorganism
Containing only 1 organism
A culture of an organism that is entirely free from all other contaminating organisms
Not contaminated by or associated with any other living organism
Pure cultures that are completely free of the presence of other organisms

14. Sterilization
Micro-organism contamination can over grow the plant culture resulting in culture death
Micro-organism contamination exhaust the nutrient media
Micro-organism can change in secondary metabolite structure or produce other compounds .

15. Source of contamination
The explant or culture
The vessels
The media
The instruments
The environment where handling is taking place

16. Aseptic Techniques Chemical treatments disinfectants, antibiotics,
sublimat
Physical treatments
heating: the most important disinfection method
electromagnetic radiation,
filtration
ultrasonic waves.

17. Disinfectans They penetrate into bacteria,
They will denature bacterial protein,
They decrease the activity of bacterial enzyme,
They inhibit bacterial growth and metabolism,
They damage the structure of cell membrane,
They change membrane permeability.

18. Disinfectans Liquid laundry bleach (NaOCl at 5-6% by vol)
Rinse thoroughly after treatment
Usually diluted 5-20% v/v in water; 10% is most common
Calcium hypochlorite – Ca(OCl)2
a powder; must be mixed up fresh each time
Ethanol (EtOH)
95% used for disinfesting plant tissues
Kills by dehydration
Usually used at short time intervals (10 sec – 1 min)
70% used to disinfest work surfaces, worker hands
Isopropyl alcohol (rubbing alcohol) is sometimes recommended

19. Antibiotics
Used only when necessary or when disinfestants are ineffective or impractical
Its use by incorporating in the media
Common antibiotics are carbenicillin, cefotaxime, rifampicin, tetracycline, streptomycin
Problems with antibiotics
tend to be selective
resistance acquisition
may obscure presence of microbes
cell/tissue growth inhibition

20. An ideal antibiotics Broad-spectrum Did not induce resistance
Selective toxicity, low side effects
Preserve normal microbial flora
BC Yang

21. Modes of action
Inhibitors of cell wall synthesis. Penicillins, cephalosporin, bacitracin, carbapenems and vancomycin.
Inhibitors of Cell Membrane. Polyenes - Amphotericin B, nystatin, and condicidin. Imidazole - Miconazole, ketoconazole and clotrimazole. Polymixin E and B.
Inhibitors of Protein Synthesis. Aminoglycosides - Streptomycin, gentamicin, neomycin and kanamycin. Tetracyclines - Chlortetracycline, oxytetracycline, doxycycline and minocycline. Erythromycin, lincomycin, chloramphenicol and clindamycin.
vancomycin
Amphotericin
Aminoglycosides
Tetracyclines
BC Yang

22. Modes of action
Inhibitors of metabolites (Antimetabolites). Sulfonamides - Sulfanilamide, sulfadiazine silver and sulfamethoxazole. Trimethoprim, ethambutol, isoniazid.
Inhibitors of nucleic acids (DNA/RNA polymerase). Quinolones - Nalidixic acid, norfloxacin and ciprofloxacin. Rifamycin and flucytosine. 
rifamycin
BC Yang

23. Sublimat (0.1 - 1%) Its activity based on Cl-
Heavy metal (Hg) denaturates proteins.
Hg is toxic for the environment, therefore recuperate the Hg-solution after use and collect in a large container.
Hg can be precipitated by adding ammonia to the solution, and siphoning the supernatant

24. UV radiation
Ultraviolet is light with very high energy levels and a wavelength of nm.
One of the most effective wavelengths for disinfection is that of 254 nm.
BC Yang

25. Heating Oven (dry heat) Microwaves (off the shelf)
Suitable for tools, containers a 160°-180° C for 3 h
Microwaves (off the shelf)
Useful for melting agar (but not gellan gum types of solidifying agents)
Special pressurized containers are required for sterilizing in a microwave
Flaming or heating of tools
Flaming – e.g., 95% EtOH in an alcohol burner is useful for sterilizing metal instruments
Bacticinerators – heats metal tools in a hot ceramic core
Heated glass beads

26. Heating
Autoclave
Steam heat under pressure (It typically generates 15 lbs/in2 and 250° F (1.1 kg/cm2 and 121° C))
It is faster and more effective
For liquids (such as water, medium), autoclave time depends on liquid volume
Recommended autoclaving times (sterilization time only):
250 ml requires 15 min
500 ml requires 20 min
1000 ml requires 25 min
Excessive autoclaving can break down organics – a typical symptom is caramelized sucrose

27. Heating Flaming or heating of tools
Flaming – e.g., 95% EtOH in an alcohol burner is useful for sterilizing metal instruments
Bacticinerators – heats metal tools in a hot ceramic core
Heated glass beads

28. Filtration Filtration of culture medium Filtration of air
Some medium ingredients are heat labile, e.g., GA, IAA, all proteins, antibiotics
Most devices use a paper cellulose filter with small pore spaces (0.22 µm)
Syringes used for small volumes, vacuum filtration for large volumes
Filtration of air
Transfer hoods usu. generate wind at linear m per min (or ft per min)
Too slow and air drops contaminants onto your work surface; too fast causes turbulence and excess filter wear
air "corridors" must be kept free of barriers to be effective

29. Sterilization Equipment

30. Sterilization Equipment
                        sterilizing paper: dry heat
                        sterilizing tools
                   laminar flow cabinet

31. Sterilization Equipment

32. Callus Culture Callus:
An un-organised mass of cells, produced when explants are cultured on the appropriate solid medium, with both an auxin and a cytokinin and correct conditions.
A tissue that develops in response to injury caused by physical or chemical means
Most cells of which are differentiated although may be and are often highly unorganized within the tissue

33. Callus formation ? Explants Callus Protoplasts Development
1. Meristems
2. Leaf sections
3. Bulb sections
4. Embryos
5. Anthers
6. Nucellus   
De-differentiation
Re-differentiation
Explants Callus
Protoplasts
Development
  Suspension cells
Organs
(leaves, roots, shoots, flowers,...) ?

34. Callus formation Stimuli :
In vivo : wound, microorganisms, insect feeding  
In vitro : Phytohormones  
1. Auxin  
2. Cytokinin  
3. Auxin and cytokinin  
4. Complex natural extracts  

35. Callus
During callus formation there is some degree of dedifferentiation both in morphology and metabolism, resulting in the lose the ability to photosynthesis.
Callus cultures may be compact or friable.
Compact callus shows densely aggregated cells
Friable callus shows loosely associated cells and the callus becomes soft and breaks apart easily.
Habituation:
The lose of the requirement for auxin and/or cytokinin by the culture during long-term culture.

36. Cell-suspension cultures
When friable callus is placed into the appropriate liquid medium and agitated, single cells and/or small clumps of cells are released into the medium and continue to grow and divide, producing a cell-suspension culture.
The inoculum used to initiate cell suspension culture should neither be too small to affect cells numbers nor too large too allow the build up of toxic products or stressed cells to lethal levels.
When callus pieces are agitated in a liquid medium, they tend to break up.

37. Cell suspension culture
Suspensions are much easier to bulk up than callus since there is no manual transfer or solid support
Cell suspension culture techniques are very important for plant biotransformation and plant genetic engineering.

38. The isolation and culture of plant protoplasts in vitro
Protoplast culture
The isolation and culture of plant protoplasts in vitro

39. Protoplast
The living material of a plant or bacterial cell, including the protoplasm and plasma membrane after the cell wall has been removed.

40. Plant Regeneration Pathways
Existing Meristems (Microcutting)
Uses meristematic cells to regenerate whole plant.
Organogenesis
Relies on the production of organs either directly from an explant or callus structure
Somatic Embryogenesis
Embryo-like structures which can develop into whole plants in a way that is similar to zygotic embryos are formed from somatic cells
(Source:Victor. et al., 2004) 40

41. Cell Differentiation Morphogenesis
The process by which cells become specialized in form and function. These cells undergo changes that organize them into tissues and organs.
Morphogenesis
As the dividing cells begin to take form, they are undergoing morphogenesis which means the “creation of form.”
Morphogenetic events lay out the development very early on in development as cell division, cell differentiation and morphogenesis overlap

42. Morphogenesis
These morphogenetic events “tell” the organism where the head and tail are, which is the front and back, and what is left and right.
As time progresses, later morphogenetic events will give instructions as to where certain appendages will be located.

43. Morphogenetic Events
Morphogenetic events, as well as cell division and differentiation, take place in all multicellular organisms.
In plants, morphogenesis and growth in overall size are not limited to embryonic and juvenile periods, they occur throughout the life of the plant.
For example, apical meristems of plants are responsible for a plant’s continued growth and development and the formation of new organs throughout the plant’s life. These are perpetually embryonic regions in the tips of shoots and roots.

46. Cloning
Using the somatic cells of a multicellular organism to generate a new organism is
Each clone is genetically identical to the parent plant.

47. Microcutting propagation
The production of shoots from pre-existing meristems only.

48. Organogenesis
The ability of non-meristematic plant tissues to form various organs de novo.
The formation of adventitious organs
The production of roots, shoots or leaves
These organs may arise out of pre-existing meristems or out of differentiated cells
This may involve a callus intermediate but often occurs without callus.

50. Indirect organogenesis
Explant
Callus
Meristemoid
Primordium

51. Direct shoot/root formation from the explant
Direct Organogenesis
Direct shoot/root formation from the explant

52. Somatic Embryogenesis
The formation of adventitious embryos
The production of embryos from somatic or “non-germ” cells.
It usually involves a callus intermediate stage which can result in variation among seedlings

53. Types of embryogenic cells
Pre-embryogenic determined cells, PEDCs
The cells are committed to embryonic development and need only to be released. Such cells are found in embryonic tissue.
Induced embryogenic determined cells, IEDCs
In majority of cases embryogenesis is through indirect method.
Specific growth regulator concentrations and/or cultural conditions are required for initiation of callus and then redetermination of these cells into the embryogenic pattern of development.

54. Various terms for non-zygotic embryos
Adventious embryos
Somatic embryos arising directly from other organs or embryos.
Parthenogenetic embryos (apomixis)
Somatic embryos are formed by the unfertilized egg.
Androgenetic embryos
Somatic embryos are formed by the male gametophyte.

55. Somatic Embryogenesis and Organogenesis
Both of these technologies can be used as methods of micropropagation.
It is not always desirable because they may not always result in populations of identical plants.
The most beneficial use of somatic embryogenesis and organogenesis is in the production of whole plants from a single cell (or a few cells).

56. Somatic embryogenesis differs from organogenesis
Bipolar structure with a closed radicular end rather than a monopolar structure.
The embryo arises from a single cell and has no vascular connection with the mother tissue.

57. Two routes to somatic embryogenesis (Sharp et al., 1980)
Direct embryogenesis
Embryos initiate directly from explant in the absence of callus formation.
Indirect embryogenesis
Callus from explant takes place from which embryos are developed.

58. Direct somatic embryogenesis
Direct embryo formation from an explant

59. Indirect Somatic Embryogenesis
Explant → Callus Embryogenic → Maturation → Germination
Calus induction
Callus embryogenic development
Multiplication
Maturation
Germination

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

61. 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)

62. Maturation
Require complete maturation with apical meristem, radicle, 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

63. 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

64. Somatic embryogenesis as a means of propagation is seldom used
High probability of mutations
The method is usually rather difficult.
Losing regenerative capacity become greater with repeated subculture
Induction of embryogenesis is very difficult with many plant species.
A deep dormancy often occurs with somatic embryogenesis

65. Peanut somatic embryogenesis

66. 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