Micropropagationand transformationprinciples, uses and

methods

By M. Sc. Student

Mohamed SalaheldinMokhtar

Under the supervision of

Prof. Dr.

Mohamed AbdelbaaethElseehy

the art and science of multiplying plants in vitro

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 …

Explant

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

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

totipotency

Micropropagationis used to

multiply novel plants

provide a sufficient number of plantlets for planting from a

stock plant which…does not

produce seeds

does not respond well to vegetative

reproduction

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

Method

Establishment

Selection of explant

Initiation and aseptic culture establishment

Selection of explant

• Explant age

• Season

• Explant size

• Plant quality

• Goal

Explant age

In many cases, older tissue will not form callus

younger tissue easier to surface disinfect

Season

dormancy

contamination rates

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.

Internal differences in hormone

balance in the tissue can result in

varying in vitro responses.

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.

GOAL

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

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

• 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.

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.

Aseptic Technique

• Killing or excluding microorganisms or their spores with heat, filters, chemicals or other sterilants

Instruments

environment

Operator

Plant material

• 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

Media

Macronutrients

Micronutrients

Carbon and energy sources

Vitamins and myo-inositol

Solidifying agents

Growth regulators

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)

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)

Carbon and energy sources

Vitamins and myo-inositol

Solidifying agents

Growth regulators

Auxins Cytokinins Gibberellinsabscisic

acidEthylene Other

Auxin and Cytokinin Ratio

Culture Initiation

Inoculation

Multiplication

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

Pretransplant

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

hardeningRooting

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.

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

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

Types of culture

(Explant base)

Seed culture

Embryo culture

Cell culture (suspension

culture)

Callus cultureBud culture

Organ culture

Protoplast culture

Meristemculture

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

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).

Methods

Direct Indirect

Direct

biolistics or particle

bombardmentelectroporation Polyethyleneglycol Microinjection

Biolistics or particle

bombardment

Electroporation

Indirect

Agrobacteriumtumefaciens

conjugation

transduction

Transformation

Infection of a plant withA. tumefaciens and

formation of crown galls

Crown Gall on Tobacco

Gene transfer in plants

Why gene transfer?• Crop improvement• Disease resistance• Stress tolerance• Improved performance• Value-added traitsBasic studies• Gene expression• Reverse genetics - understanding functioning of

unknown genes• Biochemistry and metabolism

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

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)

Transformation with Agrobacterium

• Agrobacteriumcontains a circle of DNA (Ti plasmid) that carries the desired genes

• Co-cultivation of the Agrobacterium with plant pieces transfers the DNA

Bacterial chromosomeTi Plasmid

Petri dishwith leaf pieces plus Agrobacterium

Essential Elements for Carrying a Transgene on Ti PlasmidsThe 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.

• 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.

Transfer of Modified Ti Plasmid into a PlantAgrobacterium 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.

Transgenic plant

Development of GM foods

Flavr-Savr tomato - 1st FDA approval for a food

1995 Monsanto's Roundup Ready soybeans approved for sale in the United States.

1994

First successful field trial of GM cotton- CROP1990

GM plants resistant to insects, viruses, and bacteria are field tested for the first time - USEFUL TRAITS

1985

1st transgenic plant: antibiotic resistant tobacco1983

Researchers develop the ability to isolate genes1973

First regeneration of entire plants from an in vitro culture1950