Culture media for in vitro propagation

Culture media for in vitro propagation . They constitute a fundamental element for the in vitro culture of cells, tissues and organs to achieve their development in vitro . Culture media have a series of general and specific components whose presence and concentration will depend on the objective pursued in their use. The culture media are made up of mineral substances, vitamins, amino acids, sugars, growth regulators and other elements.
Cultivation of plant cells, tissues and organs in vitro It is carried out in artificial cultivation media, which provide the necessary nutrients that the plant takes from the earth in its natural habitat and precisely the success of this type of cultivation is greatly influenced by the nature of the cultivation medium used and other environmental factors.

Summary

[ hide ]

• 1 Inorganic components of the culture medium
• 2 Organic components of the culture medium
  • 1 Carbohydrates
  • 2 Vitamins
  • 3 Amino acids and natural extracts
  • 4 Gelling agents
  • 5 Water
• 3 Plant growth regulators
• 4 Sources

Inorganic components of the culture medium

The culture media are made up of inorganic components, which are supplied in relatively large quantities (macronutrients) and others added in smaller quantities (micronutrients).
Within the macronutrients are ions of nitrogen (N), potassium (K), calcium (Ca), phosphorus (P), magnesium (Mg) and sulfur (S).
Nitrogen is added to the culture medium in the form of nitrate or ammonium ions, or the combination of both ions, magnesium sulfate (Mg SO ₄ 7H ₂O) satisfies both the magnesium and sulfur requirements, phosphorus can be added in any of the forms NaH ₂ PO ₄ .H ₂ O or KH ₂ PO ₄ , potassium is a cation that is added in the form of KCl, KNO ₃ , or KH ₂ PO ₄ , calcium is added with Ca Cl ₂ .2H ₂ O, Ca (NO ₃ ) ₂ .4H ₂ O or the anhydrous form of any salt and chlorine comes in the form of KCl or CaCl ₂ .
The micronutrients, which are added to the culture media are iron (Fe), nickel(Ni), chlorine (Cl), manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum (Mo). These elements together with carbon (C), oxygen (O) and hydrogen (H) constitute the 17 essential elements. Although these micronutrients are required in a smaller quantity, they are necessary for an adequate metabolic activity of the plant cells. Fe and Mn are essential for the synthesis of chlorophyll and the function of chloroplasts. Fe is required for the formation of chlorophyll precursors and is a component of cytochromes, ferredoxin and legohemoglobin, the latter is essential in nitrogen fixation by leguminous plants. Mn is necessary for the maintenance of the ultrastructure and the photosynthetic process. The Cu and Zn elements are required for the oxidation and hydroxylation of phenolic compounds. Zn is related to the synthesis of tryptophan, precursor of indolacetic acid (AIA) and exerts control over ribonucleases, which allows protein synthesis to be maintained in the event of environmental stress (culture medium). Cu is also a component of Plastocyanin that is essential in the functioning of the electronic transport of photosynthesis and is an activator of other enzymes such as ascorbic acid oxidase (Vitamin C), tyrosinase, laccase, phenolase and cytochromoxidase, the latter form part of the electronic transport chain of the respiratory process. Mo is part of plant nitratereductases and nitrogenases in legumes and nitrogen-fixing bacteria. Boron (B) is necessary for the maintenance of meristematic activity, it is involved in the synthesis of nitrogenous bases, in particular uracil and adenine and therefore increases the levels of cytokinins and nucleic acids. Lugo Fe is added in the form of a chelate whose molecules are capable of retaining a metal ion with various chemical bonds, forming a complex ring (a chelate) such as EDTA (ethylenedinitrotetraacetic acid), which when used in low concentrations stimulates growth by make this item available in low quantities.
The most widely used culture medium is the formulation of the Murashige and Skoog salts (1962) which was initially developed for the growth of tobacco calluses and is currently used as a basal culture medium for an important group of plants of interest to food and for ornamental purposes. Mineral salts of the culture medium of Murashige and Skoog (1962)

Organic components of the culture medium

Among the organic components of the culture medium are carbohydrates, vitamins, amino acids, natural extracts and regulators of plant growth.

Carbohydrates

The nutrition that is developed in the in vitro conditions from the different organs and tissues is widely heterotrophic with respect to carbon due to the absence or insufficiency of chlorophyll assimilation, for which it is essential to add sugars to the culture media as a source of energy and osmotic regulators. Sucrose is the sugar used universally. They are followed in importance by glucose, maltose, raffinose, fructose and galactose, among others.

Vitamins

They are necessary to carry out a series of catalytic reactions in the metabolism and are required in small quantities. The most used vitamins are:

• Thiamine (vitamin B1): it is added as thiamine hydrochloride and constitutes an essential vitamin for the growth of plant cells. It is a coenzyme of the decarboxylation of keto acids pyruvate and œ-ketoglutarate and is essential for radical growth since it is involved in the synthesis of cytokines.
• Nicotinic Acid: it is part of the coenzymes NAD and NADP that intervene in the transfer of hydrogen, in addition to being a precursor to tryptophan and therefore has a synergistic effect with AIA in the production of roots and exerts an inhibitory action in the development of axillary buds .
• Pyridoxine (Vitamin B6): It is added as pyridoxine hydrochloride (pyridoxine-HCl). It participates as a coenzyme in the metabolism of amino acids, including tryptophan, a precursor of AIA and nicotinic acid, in addition to promoting root formation.
• Myo-inositol: It is not properly a vitamin, but a sugar-alcohol. It has an effect on tissue proliferation and on the activation of organogenesis.
• Ascorbic acid and citric acid: They are added to the culture media sometimes not as vitamins but as antioxidants to avoid darkening of certain tissues.

Amino acids and natural extracts

Amino acids promote callus proliferation and organogenesis. The effects obtained by supplying amino acids seem highly variable depending on the species and the type of morphogenesis studied. Among them are glutamine, L-arginine, L-cysteine, among others.
Natural stimulating extracts are numerous products of variable and not well defined composition. Among them are yeast extract, casein hydrolyzate, peptone and coconut water (most used) among others.

Gelling agents

Agar has become the most widely used support material, providing the medium with an excellent wet gel that supports the explant. Gelrite or fitagel is also used.

Water

It is of vital importance the quality of the water used to make the culture media which must be distilled. Whenever works are carried out with tissue and cell culture in vitro, the water to be used must have the highest possible quality (deionized), and must be between 0.5 to 2 mS / cm.

Plant growth regulators

Plant growth regulators are grouped into five categories: auxins, cytokines, gibberellins, abscisic acid and ethylene. In addition to these natural plant substances, there are other products that can be used as growth regulators for in vitro cultivation .
Main growth regulators used in tissue culture.

AUXINS

• AIA: Ac. indole 3 – acetic
• ANA: Ac. naphthalenacetic
• AIB: 3-butyric indole Ac
• 4 D: Ac. 2,4 dichlorophenoxyacetic
• Picloram
• Dicamba

CYTOKININS

• BAP: 6 – benzylaminopurine
• KINETINE: 6 – furfurylaminopurine
• Z: Zeatina
• TDZ: Thidiazuron

GIBERELINES

• GA ₃: Ac. gibberellic
• GA ₁, GA ₄ , GA ₇ : (Gibberellins)

ETHYLENE ACID ABSCISICO POLYAMINS

• Putrescina
• Spermidine
• Spermine

BRASINOESTEROIDS

• Biobras 6 analogues
• Biobras 16

OLIGOSACARINES

• Pectimorf: oligogalacturonide DPS (12-14)