Plant Tissue Culture Media

1. Plant Tissue Culture Media

2. Logical Basis • For healthy and vigorous growth, intact plants need to take up from soil of an essential elements: • Relatively large amount of some inorganic elements (major plant nutrition) • a. Nitrogen (N) d. Potassium (K) • b. Calcium (Ca) e. Phosphorus (P) • c. Magnesium (Mg) f. Sulphur (S) • Small quantities of other elements (minor plant nutrient/trace elements) • a. Iron (Fe) f. Sodium (Na) • b. Chlorine (Cl) g. Manganese (Mn) • c. Zinc (Zn) h. Boron (B) • d. Copper (Cu) i. Molybdenum (Mo) • e. Nickel (Ni)

3. Essential elements (Epstein, 1971) • A plant grown in a medium adequately purged of that elements, failed to grow properly or to complete its life cycle • It is a constituent of a molecule that is known to be an essential metabolite

4. Functions of medium • Provide water • Provide mineral nutritional needs • Provide vitamins • Provide growth regulators • Provide amino acids • Provide sugars • Access to atmosphere for gas exchange • Removal of plant metabolite waste

5. Plant tissue culture media • Macronutrients (always employed) • Micronutrients (nearly always employed, although sometimes just one element, iron, has been used) • Vitamins (generally incorporated , although the actual number of compounds added, varies greatly) • Amino acids and other nitrogen supplements (usually omitted, but sometimes used with advantage) • Sugar (nearly always added, but omitted for some special purposes) • Undefined supplements (which, when used, contribute some above components, and also plant growth substances or regulants) • Buffers (have seldom been used in the past, but recently suggest that the additions of organic acids or buffers could be beneficial in some circumstances) • A solidifying agent (used when a semi solid medium is required)

6. Macronutrient • Macronutrients for plant tissue culture are provided from salt, however plant absorb entirely as ions • Nitrogen is mainly absorbed in the form of ammonium or nitrate • Phosphorus as the phosphate ions • Sulphur as sulphate ions • The most important step in deriving medium is the selection of macronutrient ions in the correct concentration and balanced • The salts normally used to provide macroelements also provide sodium and chlorine, however, plant cell tolerate high concentration of both ions without injury, these ions are frequently given little importance when contemplating media changes

7. Quantity of the Macronutrient

8. Nitrogen • It is essential to plant life • Both growth and morphogenesis is markedly influenced by the availability of nitrogen and the form in which it is presented • Most media contain more nitrate than ammonium ions. Most intact plants, tissues and organ taken up nitrogen effectively, and grow more rapidly on nutrient solutions containing both nitrate and ammonium ions • Nitrate has to be reduced to ammonium before being utilized biosynthetically • Ammonium in high concentration is latent toxic • For most type of culture, nitrate needs to be presented together with the reduced form of nitrogen and tissue will usually fail to grow on a medium with nitrate as the only nitrogen source

9. NH4+ and NO3- Regulate Medium pH and Root Morphogenesis of Rose Shoots

10. Amino acids • Amino acids can be added to satisfy the requirement for reduced nitrogen, but as they are expensive to purchase, they will only be used on media for mass propagation where this results in improved result • A casein hydrolysate, yeast extract which mainly consist of a mixture of amino acids substantially increased the yield of callus • Organic supplements have been especially beneficial for growth or morphogenesis when cells were cultured on media which do not contain ammonium ions • Glycine os an ingredient of many media. It is difficult to find hard evidence that glycine is really essential for so many tissue culture, but possible it helps to protect cell membranes from osmotic and temperature stress

11. Amino Acids • The most common sources of organic nitrogen used in culture media are amino acid mixtures. • Its uptake more rapidly than in organic amino acids . (e.g., casein hydrolysate), L-glutamine, L-asparagine, and adenine. When amino acids are added alone, they can be inhibitory to cell growth. .

12. Beneficial effects of amino acids • Rapid growth • Protoplast cell division • Conservation of ATP • AS chelating agent • Enhanced nitrogen assimilation • Not toxic as ammonium • As buffer

13. Micronutrients • Plant requirement for microelement have only been elucidated in the 19th century • In the early of 20th century, uncertainty still existed over the nature of the essential microelements • many tissue undoubtedly grown successfully because they were cultured on media prepared from impure chemicals or solidified with agar which acted as a micronutrient source • In the first instance, the advantage of adding micronutrients was mainly evaluated by their capability to improve the callus growth or root culture • Knudson (1922) incorporated Fe and Mn on very successful orchid seed media • Heller (1953) was first well demonstrated the advantages of microelement on tissue culture media

14. MS medium was formulated from the ash content of tobacco callus. The higher concentration of salts substantially enhanced cell division Quantity of the Micronutrient

15. Chelating agent • Some organic compounds are capable of forming complexes with metal cations, in which the metal is held with fairly tight chemical bonds • Metal can be bound (sequestered) by a chelating agent and held in solution under conditions where free ions would react with anions to form insoluble compounds, and some complexes can be more chemically reactive than the metals themselves • Chelating agents vary in their sequestering capacity according to chemical structure and their degree of ionisation, which changes with pH of the solution • Naturally –occurring compounds can act as chelating agents such as proteins, peptides, carboxylic acids and amino acids • There are also synthetic chelating agents with high avidity for divalent and trivalent ions

16. Chelating agents

17. Iron and iron chelates • A key properties of iron is its capacity to be oxidized easily from the ferrous (Fe(II)) to the ferric (Fe(III)) state and for ferric compounds to be readily reduced back to the ferrous form • Iron is primarily used in the chloroplasts, mitochondria and peroxisomes for effecting oxidation/reduction reaction • It is a component of ferredoxin proteins which function as electron carriers in photosynthesis • Iron is an essential micronutrient for plant tissue culture and can be taken up as either ferrous or ferric ions • Iron may not be available to plant cells, unless the pH falls sufficiently to bring free ions back to solutions • Iron can be chelated with EDTA • The addition of Fe-EDTA chelate greatly improved the availability of the element

18. Carbon Source • 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, manitol 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 • The presence of sucrose specifically inhibits chlorophyll formation and photosynthesis, making autotrophic growth less feasible • Sucrose in the culture media is usually hydrolyzed totally or partially into the component monosaccharides glucose and fructose • The general superiority of sucrose over glucose may be on account of the more effective translocation of sucrose to apical meristems

19. 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 • Thiamine (vitamin B1) Essential as a coenzyme in the citric acid cycle • Nicotinic acid (niacin) and pyridoxine (B6)

20. Organic supplement • Myo-inositol Although it is not essential for growth of many plant species, its effect on growth is significant. Part of the B complex, in phosphate form is part of cell membranes, organelles and is not essential to growth but beneficial • Complex organics Such as coconut milk, coconut water, yeast extract, fruit juices and fruit pulps.

21. Physical support agents • A. Gelling agents • When semi-solid or solid culture media are required, gelling agents are used. • An example: • Agar, agarose, gelrite, phytagel • Structural supports • Filter paper bridges, liquid permeable membrane support systems

22. Agar • Agar is the most commonly used gelling agent • It is a natural product extracted from species of red algae, especially Gelidiumamansii • It is synthetic polysaccharide gelling agents 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 (50 -90%). • 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

23. 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. • Gels are not digested by plant enzymes • Agar does not strongly react with media constituent • 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.

24. 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.

25. 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

26. 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.

27. Commercial 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

28. Hormone (the Greek word hormaein, meaning "to excite"). Small organic molecule that elicits a physiological response at very low concentrations Chemical signals that coordinate different parts of the organism Internal and external signals that regulate growth are mediated, at least in part, by growth-regulating substances, or hormones

29. Plant Hormone Plant hormones differ from animal hormones in that:  • No evidence that the fundamental actions of plant and animal hormones are the same. • Unlike animal hormones, plant hormones are not made in tissues specialized for hormone production. (e.g., sex hormones made in the gonads, human growth hormone - pituitary gland)  • Unlike animal hormones, plant hormones do not have definite target areas (e.g., auxins can stimulate adventitious root development in a cut shoot, or shoot elongation or apical dominance, or differentiation of vascular tissue, etc.). 

30. 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.

32. Auxins • Absolutely essential (no mutants known) • One compound: Indole-3-acetic acid. • Many synthetic analogues: NAA, IBA, 2,4-D, 2,4,5-T, Picloram Cheaper & more stable • Generally growth stimulatory. • Promote rooting • Stimulate cell elongation • Increase the rate of transcription • Mediate the response of bending in response to gravity or light • Produced in meristems, especially shoot meristem and transported through the plant in special cells in vascular bundles.

33. Cytokinins • Absolutely essential (no mutants known) • Natural compound: Zeatin, 2-isopentyl adenine (2iP) • Synthetic analogues: Benyzladenine (BA), Kinetin. • Stimulate cell division (with auxins). • Promotes formation of adventitious shoots • Stimulate cell division • Stimulate dark germination • Stimulate leaf expansion • Produced in the root meristem and transported throughout the plant as the Zeatin-riboside in the phloem.

34. Auxin and Cytokinin Ratio

35. Gibberellins (GA’s) • A family of over 70 related compounds, all forms of Gibberellic acid and named as GA1, GA2.... GA110. • Commercially, GA3 and GA4+9 available. • Stimulate etiolation of stems. • Help break bud and seed dormancy. • Stimulate stem elongation by stimulation cell division and elongation • Stimulate germination of pollen • Produced in young leaves

36. Abscisic Acid (ABA) • Only one natural compound. • Promotes leaf abscission and seed dormancy. • Plays a dominant role in closing stomata in response to water stress • Involved in the abscission of buds, flower and fruits • Inhibit cell division and elongation • Has an important role in embryogenesis in preparing embryos for desiccation. • Helps ensure ‘normal’ embryos.

37. Ethylene • Gas - diffuses through tissues • Stimulates abscission and fruit ripening • Used in commercial ripening for bananas & green picked fruit • Involved in leaf abscission & flower senescence • Primarily synthesized in response to stress • Regulate cell death programming

38. Brassinosteroids • Promote shoot elongating • Inhibit root growth • Promote ethylene biosynthesis • Enhance resistance to chilling, disease and herbicides

39. Salicylic acid • Promote flowering • Stimulate plant pathogenesis protein production Jasmonate Play an important role in plant defence mechanisms

40. Jasmonate • Play an important role in plant defence mechanisms

41. 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.

42. 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.

44. 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.

45. 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.

46. 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.

48. Thank you