Plant Tissue Culture

Plant Tissue Culture Dr Maged El-Sayed Mohamed memohamed@zu.edu.eg maged789@hotmail.com

Course outline • Definitions • Culture environment • Physical factors • Growth medium • Plant growth regulators • Culture types • Plant regeneration • Micropropagation

Summary of the last lecture

Definitions • Plant Tissue culture:It is the ability to in vitro generate a large number of plant cells or even the whole plant from isolated plant cells or tissue. • Plasticity:A property that allows plants to alter their metabolism, growth and development to best suit their environment • Totipotency:The ability to initiate cell division from any tissue or cell of the plant in response to particular stimuli to regenerate whole plant with maintenance of genetic potential.

The culture environment • The physical factors: • Temperature • pH • The gaseous environment • Light (quality and duration) • Osmotic pressure All these factors have to be maintained within acceptable limits for the success of plant tissue culture

Nitrogen (N) • Potassium (K) • Phosphorous (P) • Calcium (Ca) • Magnesium (Mg) • Sulfur (S) • Iron(Fe) • Manganese (Mn) • Zinc (Zn) • Boron (B) • Cupper(Cu) • Molybdenum(Mo) • Cobalt (Co) • Iodine (I)

Organic supplement • Vitamins: • Only thiamine (vitamin B1) is essential for most plant cultures, it is required for carbohydrate metabolism and the biosynthesis of some amino acids, • Myo-inositol • Although it is not essential for growth of many plant species, its effect on growth is significant. • Complex organics • Such as coconut milk, coconut water, yeast extract, fruit juices and fruit pulps.

Source of carbon • Sugars • Most plant tissue cultures are not highly autotrophic due to limitation of CO2. Therefore, sugar is added to the medium as an energy source. • Sucrose is the most common sugar added, although glucose, fructose, and sorbitol are also used in certain instances. • The concentration of sugars in nutrient media generally ranges from 20 to 40 g/l. • Sugars also contribute to the osmotic potential in the culture

Gelling agents • When semi-solid or solid culture media are required, gelling agents are used.

Agar • Agar is the most commonly used gelling agent. • Composition:Agar consists of 2 components • Agarose is an alternating D-galactose and 3,6-anhydro-L-galactose with side chains of 6-methyl-D-galactose residues. • Agaropectin is like agarose but additionally contains sulfate ester side chains and D-glucuronic acid. • Agar tertiary structure is a double helix the central cavity of which can accommodate water molecules

Advantages: • Agar is an inert component, form a gel in water that melt at 100 ° C and solidify at nearly 45 ° C • Concentrations commonly used in plant culture media range between 0.5% and 1% • If necessary, agar can be washed to remove inhibitory organic and inorganic impurities. • Disadvantages: • Agar does not gel well under acidic conditions (pH <4.5). • The inclusion of activated charcoal in media may also inhibit gelling of agar.

Agarose • It is extracted from agar leaving behind agaropectin and its sulfate groups. • It is used when the impurities of agar are a major disadvantage.

Gelrite™ • Gelrite consists of a polysaccharide produced by the bacterium Pseudomonas elodea. • It gives clear-solidified medium that leads to detection of contamination at an early stage. • Gelrite requires more stirring than agar. • Concentration of divalent cations such as calcium and magnesium must be within the range of 4-8 mM/L or the medium will not solidify

Phytagel™ • It is an agar substitute produced from a bacterial substrate composed of glucuronic acid, rhamnose and glucose. • It produces a clear, colorless, high-strength gel, which aids in detection of microbial contamination. • It is used at a concentration of 1.5-2.5 g/L. • It should be prepared with rapid stirring to prevent clumping.

Commertial Media Formulations: • Murashige and Skoog (MS) • Linsmaier and Skoog (LS) • White Medium • Gamborg medium • Schenk and Hildebrandt medium • Nitsch and Nitsch Medium • Lloyd and McCown Woody plant medium • Knudson’s medium

Plant growth regulators: Definition: Plant hormone or Phytohormone or Plant growth regulator (PGR): • They are small organic molecule that elicits a physiological response at very low concentrations.

Characteristics: • Synthesized by plants. • Show specific activity at very low concentrations • Display multiple functions in plants. • Play a role in regulating physiological phenomena in vivo in a dose-dependent manner • They may interact, either synergistically or antagonistically, to produce a particular effect.

Break

Auxin and Cytokinin Ratio

Culture Types • Explants : Sterile pieces of a whole plant from which cultures are generally initiated • 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.

Root tip: • Root cultures can be established from explants of the root tip of either primary or lateral roots. • Shoot tip: • The shoot apical meristem from either axillary or adventitious buds can be cultured in vitro. • Embryo: • Both immature and mature embryos can be used as explants to generate callus cultures or somatic embryos. • Immature, embryo-derived callus is the most popular method of monocot plant regeneration.

Haploid tissue • Male gametophyte (Pollen in anthers) or female gametophyte (the ovule) can be used as an explant. • Haploid tissue cultures can produce haploid or di-haploid plants due to doubling of chromosomes during the culture periods.

Callus: • Definition: It is an unspecialized and unorganized, growing and dividing mass of cells, produced when explants are cultured on the appropriate solid medium, with both an auxin and a cytokinin and correct conditions. • During callus formation there is some degree of dedifferentiation both in morphology and metabolism, resulting in the lose the ability to photosynthesis.

This necessitates the addition of other components e.g.: vitamins and, a carbon source to the culture medium, in addition to the usual mineral nutrients. • Habituation: it is the lose of the requirement for auxin and/or cytokinin by the culture during long-term culture. • Callus cultures may be compact or friable. • Compact callus showsdensely aggregated cells • Friable callus shows loosely associated cells and the callus becomes soft and breaks apart easily.

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. • Cell suspension culture techniques are very important for plant biotransformation and plant genetic engineering.

Summary • Gelling Agents and its types • Agar, Agarose, GilRite and Phytagel • Plant growth regulators: • Auxin, Cytokinin, Gibberelin, Abscisic and ethylene • Culture types: Callus and cell suspension cultures

Thank you

Plant Tissue Culture