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

Explants

•The smaller the explant the better the chances to overcome specific phytopathological problems (virus, microplasm, bacteria), but it decreases the survival rate

InoculumA 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 culture Bud 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 culture2. Root Root culture3. Leaf Leaf culture4. 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

Production of seedling from crop which multiply through root

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

sterilantsTissue culture is an aseptic

techniqueAseptic technique:-Sterile-Free of pathogenic microorganisms-Free from the living germs of disease and fermentation-Conditions established to exclude contaminants

Axenic cultureGermfreeUncontaminated 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 organismsPure cultures that are completely free of the presence of other organisms

Source of contamination

The explant or culture The vessels The media

The instruments The environment where handling is

taking place

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

method • electromagnetic radiation, • filtration• ultrasonic waves.

DisinfectansThey 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

AntibioticsUsed only when necessary or when

disinfestants are ineffective or impracticalIts use by incorporating in the mediaCommon antibiotics are carbenicillin,

cefotaxime, rifampicin, tetracycline, streptomycin

Problems with antibiotics• tend to be selective• resistance acquisition• Make unclear, the presence of microbes• cell/tissue growth inhibition

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

19BC Yang

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.

20

Amphotericin

Tetracyclines

Aminoglycosides

vancomycin

BC Yang

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.

21BC Yang

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 tools

Flaming – 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 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 cultureSuspensions 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 PathwaysExisting 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 EmbryogenesisEmbryo-like structures which can develop into whole plants in a way that is similar to zygotic embryos are formed from somatic cells

Cell DifferentiationThe 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 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.

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

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.

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 embryogenesisDirect embryo formation from an explant

Indirect Somatic EmbryogenesisExplant → Callus Embryogenic → Maturation →

Germination

1.Calus induction2.Callus embryogenic development

3.Multiplication4.Maturation

5.Germination

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