At the time of the early plant tissue culture experiments, uncertainty still existed over the nature of the essential microelements. Many tissues were undoubtedly grown successfully because they were cultured on media prepared from impure chemicals (see below) or solidified with agar, which acted as a micronutrient source.
In the first instance, the advantage of adding various micronutrients to culture media was mainly evaluated by the capability of individual elements to improve the growth of undifferentiated callus or isolated root cultures. Knudson (1922) incorporated Fe and Mn in his very successful media for the non-symbiotic germination of orchid seeds, and, following a recommendation by Berthelot (1934), Gautheret (1939) and Nobecourt (1937) included in their media (in addition to iron) copper, cobalt, nickel, titanium and beryllium. Zinc was found to be necessary for the normal development of tomato root systems (Eltinge and Reed, 1940), and without Cu, roots ceased to grow (Glasstone, 1947). Hannay and Street (1954) showed that Mo and Mn were also essential for root growth.
An advantage adding five micronutrients to tissue culture media was perhaps first well demonstrated by Heller in 1953 who found that carrot callus could be maintained for an increased number of passages when Fe, B, Mn, Zn and Cu were present.
Micronutrients tend to be added to modern media by the addition of fairly standard chemicals. Street (1977) rightly emphasised that even analytical grade chemicals contain traces of impurities that will provide a hidden supply of micronutrients to a medium. An illustration of this, comes from the work of Dalton et al., (1983) who found traces of silicon (Si) in a precipitate from MS medium which had been made up with analytical grade laboratory chemicals. Gelling agents contain inorganic elements but whether cultures can utilize them is unclear. Amounts of contaminating substances in chemicals would have been greater in times past, so that an early medium such as Knudson (1922; 1943) B, prepared today with highly purified chemicals, will not have quite the same composition as when it was first used by Knudson in 1922; the addition of some micronutrients might improve the results obtained from a present-day formulation of such early media.
Most modern culture media use the microelements of Gamborg et al., (1968) B5 medium, or the more concentrated mixtures in MS or Bourgin and Nitsch (1967) H media. Several research workers have continued to use Heller (1953) micro-nutrient formulation, even though higher levels are now normally recommended. Quoirin and Lepoivre
(1977) showed clearly that in conjunction with MS or their Quoirin and Lepoivre (1977) B macro-elements, the concentration of Mn in Heller's salts should be increased by 100-fold to obtain the most effective growth of Prunus meristems.
Cell growth and morphogenesis of some species may even be promoted by increasing the level of micronutrients above that recommended by Murashige and Skoog (1962). The induction and maintenance of callus and growth of cell suspensions of juvenile and mature organs of both Douglas fir and loblolly pine, was said to be improved on Litvay et al., (1981) LM medium in which Mg, B, Zn, Mo, Co and I were at 5 times the concentration of MS microelements, and Mn and Cu at 1.25 and 20 times respectively (Litvay et al., 1981; Verma et al., 1982). Other authors to have employed high micronutrient levels are Barwale et al., (1986) who found that the induction of adventitious shoots from callus of 54 genotypes of Glycine max was assisted by adding four times the normal concentration of minor salts to MS medium.
A further example of where more concentrated micro-elements seemed to promote morphogenesis is provided by the work of Wang, et al., (1980, 1981). Embryogenesis could be induced most effectively in callus derived from Hevea brasiliensis anthers, by doubling the concentration of microelements in MS medium, while at the same time reducing the level of macronutrients to 60-80% of the original.
Despite these reports, few research workers seem to have accepted the need for such high micronutrient levels. To diminish the occurrence of hyperhydricity in shoot culture of carnation, Dencso (1987) reduced the level of micronutrients (except iron, which was as recommended by Dalton et al., 1983) to those in MS medium, but this mixture was inadequate for Gerbera shoot cultures and the rate of propagation was less than that with the normal MS formulation.
The need for macronutrient concentrations to be optimised as the first step in media development seems to be emphasised by results of Eeuwens (1976). In an experiment with factorial combinations of the macro- and micro-nutrient components of four media, his Eeuwens (1976) Y3 micronutrients gave a considerable improvement in the growth of coconut callus, compared with other micro-element mixtures, when they were used with Y3 and MS macronutrients, but not when used with those of White (1942) or Heller (1953; 1955).
Welander (1977) obtained evidence, which suggested that plant cells are more demanding for minor elements when undergoing morphogenesis. Petiole explants of Begonia hiemalis produced callus on media without micronutrients, but would only produce adventitious shoot buds directly when micronutrients were added to the macronutrient formulation. The presence of iron is particularly important for adventitious shoot and root formation (Legrand, 1975).
That mineral nutrition can influence cellular differentiation in combination with growth hormones, was shown by Beasley et al., (1974). Cotton ovules cultured on a basic medium containing 5.0 mM IAA and 0.5 mM GA3, required 2 mM calcium (normally present in the medium) for the ovules to develop fibres. Magnesium and boron were essential for fibre elongation.
3.5. THE ROLES OF MICRONUTRIENTS
The essential micronutrient metals Fe, Mn, Zn, B, Cu, Co and Mo are components of plant cell proteins of metabolic and physiological importance. At least five of these elements are, for instance, necessary for chlorophyll synthesis and chloroplast function (Sundqvist et al., 1980). Micronutrients have roles in the functioning of the genetic apparatus and several are involved with the activity of growth substances.
Manganese (Mn) is one of the most important microelements and has been included in the majority of plant tissue culture media. It is generally added in similar concentrations to those of iron and boron, i.e. between 25-150 mM. Manganese has similar chemical properties to Mg2+ and is apparently able to replace magnesium in some enzyme systems (Hewitt, 1948). However there is normally 50- to 100-fold more Mg2+ than Mn2+ within plant tissues, and so it is unlikely that there is frequent substitution between the two elements.
The most probable role for Mn is in definition of the structure of metalloproteins involved in respiration and photosynthesis (Clarkson and Hanson, 1980). It is known to be required for the activity of several enzymes, which include decarboxylases, dehydrogenases, kinases and oxidases and superoxide dismutase enzymes. Manganese is necessary for the maintenance of chloroplast ultra-structure. Because Mn(II) can be oxidized to Mn(IV), manganese plays an important role in redox reactions. The evolution of oxygen during photosystem II of the photosynthetic process, is dependent on a Mn-containing enzyme and is proportional to Mn content (Mengel and Kirkby, 1982; Shkolnik, 1984). Mn is toxic at high concentration (Sarkar et al., 2004).
In tissue cultures, omission of Mn ions from Doerschug and Miller (1967) medium reduced the number of buds initiated on lettuce cotyledons. A high level of manganese could compensate for the lack of molybdenum in the growth of excised tomato roots (and vice versa) (Hannay and Street, 1954). Natural auxin levels are thought to be reduced in the presence of Mn2+ because the activity of IAA-oxidase is increased. This is possibly due to Mn2+ or Mn-containing enzymes inactivating oxidase inhibitors, or because manganous ions are one of the cofactors for IAA oxidases in plant cells (Galston and Hillman, 1961). Manganese complexed with EDTA increased the oxidation of naturally-occurring IAA, but not the synthetic auxins NAA or 2,4-D (MacLachlan and Waygood, 1956). However, Chee (1986) has suggested that, at least in blue light, Mn2+ tends to cause the maintenance of, or increase in, IAA levels within tissues by inactivating a co-factor of IAA oxidase. When the Mn2+ level in MS medium was reduced from 100 mM to 5 mM, the production in blue light, of axillary shoots by Vitis shoot cultures was increased.
Zinc is a component of stable metallo-enzymes with many diverse functions, making it difficult to predict the unifying chemical property of the element, which is responsible for its essentiality (Clarkson and Hanson, 1980). Zinc is required in more than 300 enzymes including alcohol dehydrogenase, carbonic anhydrase, superoxide dismutase and RNA-polymerase. Zinc forms tetrahedral complexes with N-, O-, and S-ligands. In bacteria, Zn is present in RNA and DNA polymerase enzymes, deficiency resulting in a sharp decrease in RNA levels. DNA polymerase is concerned with the repair of incorrectly formed pieces of DNA in DNA replication, and RNA polymerase locates the point on the DNA genome at which initiation of RNA synthesis is to take place. Divalent Mg2+, Mn2+ or Co2+ are also required for activation of these enzymes (Eichhorn, 1980).
Zinc deficient plants suffer from reduced enzyme activities and a consequent diminution in protein, nucleic acid and chlorphyll synthesis. Molybdenum-and zinc-deficient plants have a decreased chlorophyll content and poorly developed chloroplasts. Plants deprived of zinc often have short internodes and small leaves.
The concentration of Zn in MS medium is 30 ^M but amounts added to culture media have often varied widely between 0.1-70 ^M and experimental results to demonstrate the most appropriate level are limited. When Eriksson (1965) added 15 mg/l Na2ZnEDTA.2H2O (40^M Zn2+)to Haplopappus gracilis cell cultures, he obtained a 15% increase in cell dry weight which was thought to be due to the presence of zinc rather than the chelating agent. Zinc was also shown to increase growth of a rice suspension. The highest concentration tested, 520 ^M, resulted in the fastest rate of growth and it was suggested that zinc had increased auxin activity (see below) (Hossain et al., 1997). Zinc is required for adventitious root formation in Eucalyptus (Schwambach et al., 2005). In cassava, additional zinc promotes somatic embryogenesis and rooting (C.J.J.M Raemakers, pers. commun.). However, very high concentrations of zinc are found to be inhibitory, and the microelement has been noted to prevent root growth at a concentration higher than 50 ^M.
There is a close relationship between the zinc nutrition of plants and their auxin content (Skoog, 1940). It has been suggested that zinc is a component of an enzyme concerned with the synthesis of the IAA precursor, tryptophan (Tsui, 1948). The importance of Zn for tryptophan synthesis is especially noticeable in crown gall callus which normally produces sufficient endogenous auxin to maintain growth on a medium without synthetic auxins, but which becomes auxin-deficient and ceases to grow in the absence of Zn (Klein et al., 1962).
Boron is involved in plasma membrane integrity and functioning, probably by influencing membrane proteins, and cell wall intactness. Reviews have been provided by Lewis (1980) and by Blevins and Lukaszewski (1998). The element is required for the metabolism of phenolic acids, and for lignin biosynthesis: it is probably a component, or co-factor of the enzyme which converts p-coumaric acid to caffeate and 5-hydroxyferulate. Boron is necessary for the maintenance of meristematic activity, most likely because it is involved in the synthesis of N-bases (uracil in particular); these are required for RNA synthesis (Mengel and Kirkby, 1982). It is also thought to be involved in the maintenance of membrane structure and function, possibly by stabilizing natural metal chelates which are important in wall and membrane structure and function (Pollard et al., 1977; Clarkson and Hanson, 1980). Boron is concerned with regulating the activities of phenolase enzymes; these bring about the biosynthesis of phenylpropane compounds, which are polymerized to form lignin. Lignin biosynthesis does not take place in the absence of boron. Boron also mediates the action of phytochrome and the response of plants to gravity (Tanada, 1978).
Use in culture media. In the soil, boron occurs in the form of boric acid and it is this compound, which is generally employed as the source of the element in tissue cultures. Uptake of boric acid occurs most readily at acid pH, possibly in the undissociated form (Oertli and Grgurevic, 1974) or as H2BO3- (Devlin, 1975). A wide range of boron concentrations has been used in media, the most usual being between from 50 and 100 ^M: MS medium contains 100 ^M. Bowen (1979) found boron to be toxic to sugarcane suspensions above 2 mg/l (185 ^M), but there are a few reports of higher concentrations being employed (Table 3.8). High concentrations of boron may have a regulatory function; for example, 1.6-6.5 mM have been used in simple media to stimulate pollen germination (Brewbaker and Kwack, 1963; Taylor, 1972).
Boric acid reacts with some organic compounds having two adjacent cis-hydroxyl groups (Greenwood, 1973). This includes o-diphenols, hexahydric alcohols such as mannitol and sorbitol (commonly used in plant tissue culture as osmotic agents), and several other sugars, but excludes sucrose which forms only a weak association. Once the element is complexed it appears to be unavailable to plants. This led Lewis (1980) to suggest that because boric acid was required for lignin biosynthesis, vascular plants were led, during evolution, to use sucrose exclusively for the transport of carbohydrate reserves.
Although the addition of sugar alcohols and alternative sugars to sucrose can be beneficial during plant tissue culture (see Chapter 4), it is clearly necessary to return to a sucrose-based medium for long-term culture, or boron deficiency may result.
Deficiency symptoms. Boron is thought to promote the destruction of natural auxin and increase its translocation. Endogenous IAA levels increase in the absence of boron and translocation is reduced, the compound probably being retained at the site of synthesis (Goldbach and Amberger, 1986). Plants suffering from boron deficiency have restricted root systems (Odhnoff, 1957; Whittington, 1959) and a reduced capacity to absorb H2PO4- and some other ions. High levels of auxins can have the same effect on growth and ion uptake (Pollard et al., 1977). Neales (1959, 1964) showed that isolated roots stopped growing unless a minimum concentration of boron was present (although the necessity for the element was not so apparent when cultures were grown in borosilicate glass vessels). Inhibition of root elongation in the absence of boron has been shown to be due to the cessation of mitosis and the inhibition of DNA synthesis (Moore and Hirsch, 1981). Boron deficiency also results in depressed cytokinin synthesis. Cell division is inhibited in the absence of boron, apparently because there is a decrease in nuclear RNA synthesis (Ali and Jarvis, 1988). However, deficiency often leads to increased cambial growth in intact dicotyledonous plants.
Concentration of boron (^M)
Type of Culture
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