sterile pieces of a whole plant from which cultures are generally initiated explants aerial plant...

Sterile pieces of a whole plant from which cultures are generally initiated

Explants

•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

Inoculum

A subculture of plant material which is already in culture

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.

Types of explant

Types of culture (Explant base)

Plant tissue culture

Embryo culture Seed culture Meristem culture

Protoplast cultureCell culture (suspension culture)

Callus cultureBud culture

Organ culture

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

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

Embryo culture Growing embryo

aseptically in vitro on artificial nutrient media

Overcoming seed dormancy and self-sterility of seeds

Study embryo development

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

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

Root organ culture

1.Production of seedling from crop which multiply through root

2.Production of secondary metabolite

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

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

SterilizationKilling or excluding microorganisms or their spores with heat, filters, chemicals or other

sterilants

Tissue culture is an aseptic techniqueAseptic 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

Axenic culture

GermfreeUncontaminated Free from germs or pathogenic organisms Free from other microorganism Containing only 1 organismA culture of an organism that is entirely free from all other contaminating organismsNot contaminated by or associated with any other living organism Pure cultures that are completely free of the presence of other organisms

Sterilization

1. Micro-organism contamination can over

grow the plant culture resulting in

culture death

2. Micro-organism contamination exhaust

the nutrient media

3. Micro-organism can change in secondary

metabolite structure or produce other

compounds .

Source of contamination

The explant or culture

The vessels

The media

The instruments

The environment where handling is

taking place

Aseptic Techniques

Chemical treatments • disinfectants, • antibiotics, • sublimatPhysical treatments• heating: the most important disinfection

method • electromagnetic radiation, • filtration• ultrasonic waves.

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.

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

Antibiotics

Used only when necessary or when disinfestants are ineffective or impractical

Its use by incorporating in the mediaCommon 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

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An ideal antibiotics

Broad-spectrum Did not induce resistance Selective toxicity, low side effects Preserve normal microbial flora

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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.

Amphotericin

Tetracyclines

Aminoglycosides

vancomycin

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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

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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

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UV radiation Ultraviolet is light

with very high energy levels and a wavelength of 200-400 nm.

One of the most effective wavelengths for disinfection is that of 254 nm.

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Heating

•Oven (dry heat) 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 toolsFlaming – e.g., 95% EtOH in an alcohol burner is useful for

sterilizing metal instrumentsBacticinerators – heats metal tools in a hot ceramic coreHeated glass beads

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 effectiveFor liquids (such as water, medium), autoclave time depends on liquid volume Recommended autoclaving times (sterilization time only):

250 ml requires 15 min500 ml requires 20 min1000 ml requires 25 min

Excessive autoclaving can break down organics – a typical symptom is caramelized sucrose

Heating

• Flaming or heating of toolsFlaming – e.g., 95% EtOH in an alcohol burner is useful for sterilizing metal instrumentsBacticinerators – heats metal tools in a hot ceramic coreHeated glass beads

Filtration

– Filtration of culture medium•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 27-30 linear m per min (or 90-100 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

Sterilization Equipment

sterilizing paper: dry heat

sterilizing tools

laminar flow cabinet

Sterilization Equipment

Sterilization Equipment

Callus CultureCallus:

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

Explants Callus  

Protoplasts   Development   Suspension cells  

Organs  

(leaves, roots, shoots, flowers,...)  

De-differentiation Re-differentiation

1. Meristems  2. Leaf sections  3. Bulb sections  4. Embryos  5. Anthers  6. Nucellus   

Callus formation

Stimuli :

In vivo : wound, microorganisms, insect feeding  

In vitro : Phytohormones   1. Auxin   2. Cytokinin   3. Auxin and cytokinin   4. Complex natural extracts  

Callus formation

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 cellsFriable 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.

•

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.

Cell-suspension cultures

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.

Protoplast culture

The isolation and culture of plant protoplasts in vitro

Protoplast

The living material of a plant or bacterial cell, including the protoplasm and plasma membrane

after the cell wall has been removed.

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)(Source:Victor. et al., 2004)

Cell Differentiation

The process by which cells become specialized in form and function. These cells undergo changes

that organize them into tissues and organs.

MorphogenesisAs 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

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.

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.

Cloning

• Using the somatic cells of a multicellular organism to generate a new organism is

• Each clone is genetically identical to the parent plant.

Microcutting propagationThe production of shoots from pre-

existing meristems only.

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.

Indirect organogenesis

Explant

Callus

Meristemoid

Primordium

Direct OrganogenesisDirect shoot/root formation from the

explant

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

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.

Various terms for non-zygotic embryos

Adventious embryosSomatic embryos arising directly from other organs or embryos.

Parthenogenetic embryos (apomixis) Somatic embryos are formed by the unfertilized egg.

Androgenetic embryosSomatic embryos are formed by the male gametophyte.

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).

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.

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.

Direct somatic embryogenesis

Direct embryo formation from an explant

Indirect Somatic Embryogenesis

Explant → Callus Embryogenic → Maturation → Germination

1.Calus induction2.Callus embryogenic development

3.Multiplication4.Maturation5.Germination

Induction• Auxins required for induction

–Proembryogenic masses form–2,4-D most used–NAA, dicamba also used

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)

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

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– Woody plants

Somatic embryogenesis as a means of propagation is

seldom usedHigh 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

Peanut somatic embryogenesis

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