micropropagation and transformation principles, uses and methods

1. Micropropagation
and transformation
principles, uses and
methods

2. By M. Sc. Student
Mohamed Salaheldin
Mokhtar
Under the supervision of
Prof. Dr.
Mohamed Abdelbaaeth
Elseehy

5. the art and science
of multiplying
plants in vitro

6. 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 …

7. Explant
Cell, tissue or organ
of a plant that is
used to start in
vitro cultures.

8. totipotency
The capacity of a
cell (or a group of
cells) to give rise
to an entire
organism.

9. multiply
novel plants
Micropropagation
is used to
provide a sufficient number
of plantlets for planting from a
stock plant which…
does not
produce seeds
does not respond
well to vegetative
reproduction

10. MICRO. Vs MACRO.
PROPAGATION
• Small propagule
• Aseptic conditions
• Controlled
environment
• Heterotrophic growth
• Rapid multiplication
• Greater initial costs
• Larger propagule
• Non-aseptic conditions
• Less environmental
control
• Photoautotrophic
growth
• Slower multiplication
• Nominal costs

11. Method

12. Establishment
Selection of
explant
Initiation and
aseptic culture
establishment

14. Selection of explant
• Explant age
• Season
• Explant size
• Plant quality
• Goal

15. Explant age

16. In many cases, older
tissue will not form
callus
younger tissue
easier to surface
disinfect

17. Season

18. dormancy

19. contamination rates

20. EXPLANT SIZE
• the smaller the explant, the harder it is to
culture.
• The larger explants probably contain more
nutrient reserves and plant growth regulators
to sustain the culture.

21. Internal differences
in hormone
balance in the
tissue can result in
varying in vitro
responses.

22. PLANT QUALITY
• It is advisable to obtain explants from plants
which are healthy as compared to plants
under nutritional or water stress or plants
which are exhibiting disease symptoms.

23. GOAL
• Depending on what type of a response is
desired from the cell culture, the choice of
explant tissue will vary.

24. • Any piece of plant tissue can be used as an
explant

25. • For example, if clonal propagation is the goal,
then the explant will usually be a lateral or
terminal bud or shoot.
• For callus induction, pieces of the cotyledon,
hypocotyl, stem, leaf, or embryo are usually
used.
• Excellent explants for callus induction are
seedling tissues from aseptically germinated
seeds or immature inflorescences.
• Leaf tissue from the aseptically germinated seed
is a good source of tissue for protoplast
isolation.
• To produce haploid plants or callus, the anther
or pollen is cultured.

27. Initiation and aseptic culture
establishment
• The explant is surface sterilized before being
placed on the medium. Small amounts of
plant growth regulators may be added to the
medium for quick establishment of the
explant.

28. Aseptic Technique
• Killing or excluding microorganisms or their
spores with heat, filters, chemicals or other
sterilants

29. Instruments

31. environment

33. Operator

34. Plant material

36. • Liquid laundry bleach (NaOCl at 5-6% by vol)
1.Rinse thoroughly after treatment
2.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)
1.95% used for disinfesting plant tissues
2.Kills by dehydration
3.Usually used at short time intervals (10 sec – 1 min)
4.70% used to disinfest work surfaces, worker hands
• Isopropyl alcohol (rubbing alcohol) is sometimes
recommended

37. 




38. Macronutrients
Media
Micronutrients
Carbon and
energy sources
Vitamins and
myo-inositol
Growth
regulators
Solidifying
agents

39. Macronutrients
1. Carbon (C)
2. Nitrogen (N)
3. Potassium (K)
4. Hydrogen (H)
5. Calcium (Ca)
6. Phosphorus (P)
7. Oxygen (O)
8. Magnesium (Mg)
9. Sulphur (S)

40. Micronutrients
1. Iron (Fe)
2. Sodium (Na)
3. Chlorine (Cl)
4. Manganese (Mn)
5. Zinc (Zn)
6. Boron (B)
7. Copper (Cu)
8. Molybdenum (Mo)
9. Nickel (Ni)

41. Carbon and energy sources

42. Vitamins and
myo-inositol

43. Solidifying agents

44. Growth
regulators
Auxins Cytokinins Gibberellins
abscisic
acid
Ethylene Other

46. Auxin and Cytokinin Ratio

47. Culture Initiation
Inoculation

48. Multiplication
• is the taking of tissue samples produced
during the first stage and increasing their
number

52. Pretransplant
Rooting hardening
• treating the plantlets/shoots produced to
encourage root growth and "hardening." It is
performed in vitro, or in a sterile "test tube"
environment

54. Transfer from culture to the natural
environment or acclimatization
• the plantlets are removed from the plant
media and transferred to soil or (more
commonly) potting compost for continued
growth by conventional methods.

58. Advantages
Rapid & efficient propagation
Year-round production
Reduce stock plant space
Long-term germplasm storage
Production of difficult-to-propagate species
Production of disease-free plants

59. Disadvantages
X Equipment/facility intensive operation
X Technical expertise in management positions
X Protocols not optimized for all species
X Liners may not fit industry standard
X It may be too expensive

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

61. Applications of Tissue Culture
1. Embryo culture
2. Meristem culture
3. Micropropagation
4. Somatic embryogenesis and Organogenesis
5. Somaclonal variation and in vitro selection
6. Anther culture Haploid & Dihaploid Production
7. Protoplast culture (In vitro hybridization –
Protoplast Fusion)
8. Germplasm preservation

64. Plant Genetics Transformation
• transformation is the genetic alteration of a
cell resulting from the direct uptake and
incorporation of exogenous genetic material
(exogenous DNA) from its surroundings and
taken up through the cell membrane(s).

66. Methods
Direct Indirect

68. Direct
biolistics or
particle
bombardment
electroporation Polyethyleneglycol Microinjection

69. Biolistics or
particle
bombardment

71. Electroporation

72. Indirect
Agrobacterium
tumefaciens

73. conjugation
transduction

74. Transformation

75. Crown Gall on
Tobacco
Infection of a plant with
A. tumefaciens and
formation of crown galls

78. Gene transfer in plants
Why gene transfer?
• Crop improvement
• Disease resistance
• Stress tolerance
• Improved performance
• Value-added traits
Basic studies
• Gene expression
• Reverse genetics - understanding functioning of
unknown genes
• Biochemistry and metabolism

79. Plant transformation with the Ti plasmid
of Agrobacterium tumefaciens
• A. tumefaciens is a gram-negative soil bacterium which
naturally transforms plant cells, resulting in crown gall (cancer)
tumors
• Tumor formation is the result of the transfer, integration and
expression of genes on a specific segment of A. tumefaciens
plasmid DNA called the T-DNA (transferred DNA)
• The T-DNA resides on a large plasmid called the Ti (tumor
inducing) plasmid found in A.tumefaciens

80. Overview of requirements for plant
genetic transformation
• Trait that is encoded by a single gene
• A means of driving expression of the gene in
plant cells (Promoters and terminators)
• Means of putting the gene into a cell
(Vector)
• A means of selecting for transformants
• Means of getting a whole plant back from
the single transformed cell (Regeneration)

82. Transformation with Agrobacterium
• Agrobacterium
Bacterial
contains a circle of
DNA (Ti plasmid) that
carries the desired
genes
• Co-cultivation of the
Agrobacterium with
plant pieces transfers
the DNA
Ti Plasmid chromosome
Petri dish
with leaf pieces
plus Agrobacterium

84. Essential Elements for Carrying a Transgene on Ti Plasmids
The T-DNA segment contains both a transgene and a selective
marker or reporter gene. These have separate promoters and
termination signals. The marker or reporter gene must be
expressed all the time, whereas the transgene is often
expressed only in certain tissues or under certain
circumstances and usually has a promoter that can be induced
by appropriate signals.

85. • Selected single cells from the callus can be
treated with a series of plant hormones, such
as auxins and gibberellins, and each may
divide and differentiate into the organized,
specialised, tissue cells of an entire plant. The
new plant that originated from a successfully
shot cell have new genetic (heritable) traits.

86. Transfer of Modified Ti Plasmid into a Plant
Agrobacterium carrying a Ti plasmid is added to plant tissue growing in
culture. The T-DNA carries an antibiotic resistance gene (neomycin in this
figure) to allow selection of successfully transformed plant cells. Both
callus cultures (A) and liquid cultures (B) may be used in this procedure.

87. Transgenic plant

89. Development of GM foods
1950 First regeneration of entire plants from an in vitro culture
1973 Researchers develop the ability to isolate genes
1983 1st transgenic plant: antibiotic resistant tobacco
1990 First successful field trial of GM cotton- CROP
Flavr-Savr tomato - 1st FDA approval for a food
1995 Monsanto's Roundup Ready soybeans approved for
sale in the United States.
1994
GM plants resistant to insects, viruses, and bacteria are
field tested for the first time - USEFUL TRAITS
1985