H H

Allantoic acid

Fig. 3.1 The structures of allantoin and allantoic acid.

Most media contain more nitrate than ammonium ions, but as plant tissue culture media are usually not deliberately buffered, the adopted concentrations of ammonium and nitrate ions have probably been more due to practical pH control, than to the requirement of the plant tissues for one form of nitrogen or another (see chapter 4). Uptake of nitrate only takes place effectively in an acid pH, but is accompanied by extrusion of anions from the plant, leading to the medium gradually becoming less acid. By contrast, uptake of ammonium results in the cells excreting protons (H+) into the medium, making it more acid. The exchange of ions preserves the charge balance of the tissues and may also assist in the disposal of an excess of protons or hydroxyl (OH) ions generated during metabolism (Raven, 1986). Uptake of nitrogen by cell suspension cultures of Nicotiana tabacum is an active (energy-dependent) process (Heimer and Filner, 1971) and is dependent on a supply of oxygen (Buwalda and Greenway, 1989).

Plant culture media are usually started at pH 5.45.8. However, in one containing both nitrate and ammonium ions, a rapid uptake of ammonium into plant tissue causes the pH to fall to ca. 4.2-4.6. As this happens, further ammonium uptake is inhibited, but uptake of nitrate ion is stimulated, causing the pH to rise again. In unbuffered media, efficient nitrogen uptake can therefore depend on the presence of both ions. Unless otherwise stated, comments in this section on the roles of nitrate and ammonium refer to observations on unbuffered media.

There is generally a close correlation in tissue cultures between uptake of nitrogen, cell growth and the conversion of nitrogen to organic materials. A readily available supply of nitrogen seems to be important to maintain cultured cells in an undifferentiated state. The depletion of nitrogen in batch cultures, triggers an increase in the metabolism of some nitrogen-free compounds based on phenylpropanes (such as lignin), which are associated with the differentiation of secondarily-thickened cells (Hahlbrock, 1974). However, the growth in culture of differentiated cotton fibres composed largely of cellulose, is nitrate-dependent; the presence of some reduced nitrogen in the culture medium decreased the proportion of cultured embryos which produced fibres, and particularly in the absence of boron, promoted the cells of the embryos to revert to callus formation (Birnbaum et al., 1974).

Nitrate ions are an important source of nitrogen for most plant cultures, and nearly all published media provide the majority of their available nitrogen in this form. However, once within the cell, nitrate has to be reduced to ammonium before being utilised biosynthetically. Why not simply supply nitrogen as NH4+ and avoid the use of NO3- altogether? The reason lies in the latent toxicity of the ammonium ion in high concentration, and in the need to control the pH of the medium.

Conversion of nitrate to ammonium is brought about firstly by one, or possibly two, nitrate reductase enzymes, which reduce NO3- to nitrite (NO2-). One nitrate reductase enzyme is thought to be located in the cytoplasm, while the second may be bound to membranes (Nato et al., 1990). The NO2- produced by the action of nitrate reductase is reduced to NH4+ by a nitrite reductase enzyme located in plastids (Fig. 3.2). Reduction of nitrate to ammonia requires the cell to expend energy. The ammonium ions produced are incorporated into amino acids and other nitrogen-containing compounds. Nitrate and nitrite reductase enzymes are substrate induced, and their activity is regulated directly by the level of nitrate-nitrite ions within cultured cells (Chroboczek-Kelker and Filner, 1971; Hahlbrock, 1974), but also apparently by the products of the assimilation of reduced nitrogen (see below).

Fig. 3.2 The metabolism of nitrate and ammonium ions.

Unlike the ammonium ion, nitrate is not toxic and, in many plants, much is transported to the shoots for assimilation. On the other hand, the nitrite ion can become toxic should it accumulate within plant tissues or in the medium, for example when growth conditions are not favourable to high nitrite reductase activity and when nitrate is the only nitrogen source (Jordan and Fletcher, 1979; Grimes and Hodges, 1990). In Pinus pinaster, nitrate reductase is induced by the presence of KNO3, and plants regenerated in vitro exhibit an ability to reduce nitrate similar to that of seedlings (Faye et al., 1986).

For most types of culture, the nitrate ion needs to be presented together with a reduced form of nitrogen (usually the NH4+ ion), and tissues may fail to grow on a medium with NO3- as the only nitrogen source (Hunault, 1985).

2.1.3. Reduced nitrogen

In the natural environment and under most cropping conditions, plant roots usually encounter little reduced nitrogen, because bacteria rapidly oxidize available sources (Hiatt, 1978). An exception is forest soils in mountainous regions of the northern hemisphere, where nitrates are not usually available (Durzan, 1976). If NH4+, and other reduced nitrogen compounds are available, (and this is particularly the case in the aseptic in vitro environment), they can be taken up and effectively utilized by plants. In fact the uptake of reduced nitrogen gives a plant an ergonomic advantage because the conversion of nitrate to ammonium ions (an energy-requiring process) is not necessary. The free ammonium ion can cause toxicity, which, at least in whole plants, can lead to an increase in ethylene evolution (Barker and Corey, 1987; Corey and Barker, 1987). Shoots grown on an unbuffered medium containing a high proportion of ammonium ions may become stunted or hyperhydric. These effects can sometimes be reversed by transfer to a medium containing a high proportion of NO3 or to one where NO3 is the only N source (Mott et al., 1985). Hyperhydricity is the in vitro formation of abnormal organs, which are brittle and have a water-soaked appearance.

Growth of plant cultures may also be impaired in media containing high concentrations of NH4+ even when high concentrations of NO3 are present at the same time. Growth inhibition may not only be due to depressed pH (Mott et al., 1985), but may reflect a toxicity induced by the accumulation of excess ammonium ions. In normal circumstances the toxic effect of ammonium is avoided by conversion of the ion into amino acids. There are two routes by which this takes place (Fig. 3.2), the most important of which, under normal circumstances, is that by which L-glutamic acid is produced from glutamine through the action of glutamine synthetase (GS) and glutamate synthetase (GOGAT) enzymes. Compounds, which block the action of GS can be used as herbicides (De Greef et al., 1989). The reaction of a-ketoglutaric acid with NH4+ is usually less important, but seems to have increased significance when there is an excess of ammonium ions (Furuhashi and Takahashi, 1982). Detoxification and ammonium assimilation may then be limited by the availabilty of a-ketoglutaric acid, but this may be increased in vitro by adding to the medium one or more acids which are Krebs' (tricarboxylic acid) cycle intermediates. Their addition can stimulate growth of some cultures on media containing high levels of NH4+ (Gamborg, 1970).

In comparison with media having only nitrate as the nitrogen source, the presence of the ammonium ion in media usually leads to rapid amino acid and protein synthesis, and this takes place at the expense of the synthesis of carbohydrate compounds. This diversion of cellular metabolism can be disadvantageous in some shoot cultures, and can contribute towards the formation of hyperhydric shoots. Hyperhydricity no longer occurs when NH4+ is eliminated from the medium or greatly reduced. It is possible that adding an organic acid to the medium might also alleviate the symptoms on some plants.

A supply of reduced nitrogen in addition to nitrate, appears to be beneficial for at least two processes involved with cell division:

• the formation of a cell wall. Without a complete cell wall, protoplasts require a factor capable of inducing wall formation. Freshly-isolated protoplasts may contain sufficient of this substance to promote wall formation for just a few divisions. The wall-forming factor is only effective when NH4+ is present in the medium (Meyer and Abel, 1975a,b): glutamine does not substitute for NH4+:

• the activity of growth regulators. (see below) There are several reports in the literature that, with constant amounts of NO3-, ammonium sulphate has not provided such a good source of NH4+ as ammonium nitrate or ammonium chloride (De Jong et al., 1974; Steward and Hsu, 1977; Singh, 1978; Kamada and Harada, 1979). Possibly the reason is that a medium containing ammonium sulphate has a greater tendency to become acid (Harris, 1956), than one containing less sulphate ions. This would result if the presence of sulphate ions accelerated the uptake of NH4+, or slowed the uptake of NO3-. Ammonium sulphate has been used as the only source of the ammonium ion in some media used for the culture of legumes, including B5 (Gamborg et al., 1968).

2.1.4. Ammonium as the sole nitrogen source pH adjustment. If plant tissues are presented with a medium containing only NH4+ nitrogen, the pH falls steadily as the ion is taken up (for example, a decrease of 0.9 pH units in 15 days in Asparagus callus - Hunault (1985) or 0.7 pH units with potato shoots - Avila et al., (1998). Growth and morphogenesis is possible in suspension cultures containing only NH4+ ions, providing the pH of the medium is frequently adjusted by the addition of a base (Martin et al., 1977), or the medium is buffered (see below). In wild carrot, the induction of embryogenesis required the medium to be adjusted to pH 5.4 at 8 hourly intervals (Dougall and Verma, 1978). Without adjustment, the pH of media containing only NH4+ falls rapidly to a point where cells cannot grow (Dougall, 1981).

Buffering. Ammonium can also serve as the only nitrogen source when the medium is buffered (see the section on pH, below). Tobacco cells could be grown on a medium containing NH4+ nitrogen if the organic acid ion, succinate, was added to the medium. Gamborg and Shyluk (1970) found that cultured cells could be grown without frequent pH adjustment on a medium containing only NH4+ nitrogen, when a carboxylic acid was present. The organic acids appeared to minimize the acidification of the medium through NH4+ uptake. Similarly Asparagus internode callus grew just as well on NH4+ as the only nitrogen source as on a medium containing both NH4+ and NO3-, but only when organic acids (such as citrate, or malate) or MES buffer were added to the medium. When media were buffered with MES, the best callus growth occurred when the pH was 5.5 (Hunault, 1985).

The additional effect of organic acids.

Although Krebs' cycle organic acids can act as buffers, they may also act as substrates for amino acid synthesis from NH4+. To be assimilated into amino acids via the GDH enzyme, the ammonium ion must react with a-ketoglutaric acid, which is produced by the Krebs' cycle (Fig 3.2). Its availability may govern the rate at which ammonium can be metabolised by this route. The rate of assimilation might be expected to be improved by supplying the plant with a-ketoglutarate directly, or by supplying acids which are intermediates in the Krebs' cycle (citrate, iso-citrate, succinate, fumarate or malate), for then the natural production of a-ketoglutarate should increase (Gamborg, 1970).

This hypothesis was confirmed by Behrend and Mateles (1976) who concluded that succinate, or other Krebs' cycle acids, acted mainly as a nutrient, replacing a-ketoglutarate as it was withdrawn from the cycle during NH4+ metabolism and amino acid synthesis. Depletion of a-ketoglutarate causes the cycle to cease unless it, or another intermediate, is replaced. The optimum molar ratio of NH4+ to succinate, was 1.5 (e.g. 10 mM NH4+: 15 mM succinate). Chaleff (1983) thought that the growth of rice callus on Chaleff (loc. cit.) R3 (NH4) medium, containing 34 mM of only ammonium nitrogen, [Chaleff (1983) R3 NH4 medium] was enabled by the presence of 20 mM succinate or a-ketoglutarate, partly by the buffering capacity of the acids, and partly by their metabolism within the plant, where they may serve as substrate for amino acid synthesis. Similar conclusions have been reached by other workers (e.g. Fukunaga et al., (1978); Dougall and Weyrauch (1980); Hunault (1985); Molnar (1988b), who have found that compounds such as ammonium malate and ammonium citrate are effective nitrogen sources.

Orange juice promotes the growth of Citrus callus. Einset (1978) thought that this was not due to the effect of citric acid, but Erner and Reuveni (1981) showed that citric acid, particularly at concentrations above the 5.2 mM found in the juice used by Einset, does indeed promote the growth of Citrus callus; it had a more pronounced effect than other Krebs' cycle acids, perhaps due to the distinctive biochemistry of the genus.

Organic acids not only enhance ammonium assimilation when NH4+ provides the only source of nitrogen, but may sometimes also do so when nitrate ions are in attendance. The weight of rice anther callus was increased on Chaleff (1983) R3 medium, if 20 mM succinate [Chaleff (1983) R3 Succ. medium] or a-ketoglutarate was added (Chaleff, 1983). Similarly the rate of growth of Brassica nigra suspensions on MS medium, was improved either by adding amino acids, or 15 mM succinate. An equivalent improvement (apparently due entirely to buffering) only occurred through adding 300 mM MES buffer (Molnar, 1988b). However, the presence of organic acids may be detrimental to morphogenesis. In Chaleff's experiment, the presence of succinate in R3 medium markedly decreased the frequency of anther callus formation.

Photosynthesis. Although plants grown on nutrient solutions containing only NH4+ nitrogen have been found to possess abnormally high levels of PEP enzyme (Arnozis et al., 1988) (the enzyme facilitating CO2 fixation in photosynthesis), media containing high levels of NH4+ tend to inhibit chlorophyll synthesis (Yoshida and Kohno, 1982) and photosynthesis.

Plants are able to absorb urea, but like the ammonium ion, it is not a substance that is normally available in soils in the natural environment. It is however produced as a by-product of nitrogen metabolism; small quantities are found in many higher plants, which are able to utilise urea as a source of nitrogen, providing it is first converted to ammonium ions by the enzyme urease. In legumes and potato, urease requires the microelement nickel for activity (see below). In conifers, the epidermal cells of cotyledons and cotyledons are capable of urease induction and ammonium ion formation (Durzan, 1987).

Urea can be used as the sole nitrogen source for cultures, but growth is less rapid than when ammonium and nitrate ions are supplied (Kirkby et al., 1987); urease enzyme increases after cultures have been maintained for several passages on a urea-based medium (King, 1977; Skokut and Filner,

1980). Although the metabolism of urea, like that of other reduced nitrogen compounds, causes the production of excess hydrogen ions, less are predicted to be secreted into the medium than during the utilisation of NH4+ (Raven, 1986), so that urea is less suitable than ammonium to balance the pH of media containing NO3-. Nitrate ions are utilized in preference to urea when both nitrogen sources are available (King, 1977). Urea is able to serve as a reduced nitrogen source during embryogenesis (Durzan, 1987), but has been used in relatively few culture media, and of these, none has been widely adopted (George et al., 1987).

2.1.6. Media with nitrate and ammonium ions

Most intact plants, tissues and organs take up nitrogen more effectively, and grow more rapidly, on nutrient solutions containing both nitrate and ammonium ions, than they do on solutions containing just one of these sources. Although in most media, reduced nitrogen is present in lower concentration than nitrate, some morphogenic events depend on its presence, and it can be used in plant cultures in a regulatory role. Adventitious organs may also develop abnormally if NH4+ is missing (Drew, 1987).

Possible explanations, which have been put forward for the regulatory effect of NH4+ are:

• that the reduction and assimilation of NO3 is assisted by the presence of of NH4+ or the products of its assimilation (Bayley et al., 1972a,b; Mohanty and Fletcher, 1978; 1980). When grown on a medium containing a small amount of NH4+ nitrogen in addition to nitrate, suspension cultured cells of 'Paul's Scarlet' rose accumulated twice as much protein as when grown on a medium containing only nitrate, even though ammonium finally accounted for only 10% of the total protein nitrogen (Mohanty and Fletcher, 1980). Dougall (1977) considered this to be an oversimplified interpretation, moreover nitrate reductase activity is effectively increased by the presence of NO3 (Müller and Mendel, 1982) and in some plants, a high concentration of NH4+ inhibits nitrate reductase activity (see below).

• that ammonium ions effectively buffer plant nutrient media in the presence of nitrate and so enhance nitrate uptake (see the section on pH).

Cultures of some plants are capable of growing with only NO3 nitrogen (e.g. cell cultures of Reseda luteoli, soybean, wheat, flax and horse radish -Gamborg (1970); callus of Medicago sativa - Walker and Sato (1981), although yields are generally better when the medium is supplemented with NH4+.

Craven et al., (1972) with carrot, and Mohanty and Fletcher (1978) with Rosa 'Paul's Scarlet', found that the presence of NH4+ was particularly important during the first few days of a suspension culture. After that cells increase in cell number and dry weight more rapidly on NO3- nitrogen alone.

The response of plant cultures to nitrate and ammonium ions depends to a large extent on the enzymes shown in Fig 3.2, and the manner in which their activities are increased or inhibited in different tissues by the presence of the ions. These factors vary according to the degree of differentiation of the tissue (Suzuki and Nato, 1982), its physiological age, and its genotype. For example, the high level of NH4+ in MS medium inhibited the activity of glutamate synthetase enzyme in soybean suspension cultures (Gamborg and Shyluk, 1970), while in N. tabacum, a peak of glutamate dehydrogenase (GDH) appeared to exist at 10 mM NH4+ (Lazar and Collins, 1981). The activity of GDH and NADH-dependent GOGAT developed rapidly in cultured tobacco cells, while nitrate reductase and ferridoxin-dependent GOGAT activity increased more slowly during growth. By contrast, in sunflower cultures, the specific activity of GDH and ferridoxin-dependent GOGAT only reached a maximum at the end of growth, and the presence of 15 mM NH4+ inhibited the activity of nitrate reductase, indicating that the cells were entirely dependent on the reduced nitrogen in the medium (Lenee and Chupeau, 1989).

In consequence of such variation, the relative concentrations of ammonium and nitrate in media may need to be altered for different cultures.

2.1.7. The correct balance of ions

When trying to find media formulations suitable for different plant species and different kinds of cultures, two important factors to be considered are:

• the total concentration of nitrogen in the medium;

• the ratio of nitrate to ammomium ions.

There is a high proportion of NH4+ nitrogen in MS medium [ratio of NO3" to NH4+, 66:34] and the quantity of total nitrogen is much higher than that in the majority of other media. For some cultures, the total amount of nitrogen is too high and the balance between the two forms in the medium is not optimal.

It is noticeable that in several media used for legume culture, there is a greater proportion of NO3 to NH4+ ions than in MS medium. Evans et al., (1976) found that soybean leaf callus grew more rapidly and formed more adventitious roots when the ammonium nitrate in MS medium was replaced with

500 mg/l ammonium sulphate. A reduction in the ammonium level of their medium for the callus culture of red clover, was also found to be necessary by Phillips and Collins (1979, 1980) to obtain optimum growth rates of suspension cultured cells. A similar adjustment can be beneficial in other genera. Eriksson (1965) was able to enhance the growth rate of cell cultures of Haplopappus gracilis when he reduced the ammonium nitrate concentration to 75% of that in MS medium, and doubled the potassium dihydrogen phosphate level.

Table 3.4 lists examples of where changes to the nitrogen content of MS medium resulted in improved in vitro growth or morphogenesis. It will be seen that the balance between NO3 and NH4+ in these different experiments has varied widely. This implies that the ratio between the two ions either needs to be specifically adjusted for each plant species, or that the total nitrogen content of the medium is the most important determinant of growth or morphogenesis. Only occasionally is an even higher concentration of nitrate than that in MS medium beneficial. Trolinder and Goodin (1988) found that the best growth of globular somatic embryos of Gossypium was on MS medium with an extra 1.9 g/l KNO3.

There are reports that adjustments to the nitrogen content and ratio of NO3 to NH4+, can be advantageous in media containing low concentrations of salts (Table 3.5). Biedermann (1987) found that even quite small adjustments could be made advantageously to the NO3 content of a low salt medium [that of Biedermann (1987)] for the shoot culture of different Magnolia species and hybrids, but too great a proportion of NO3 was toxic.

2.1.8. The nitrate-ammonium ratio for various purposes

Root growth. Root growth is often depressed by NH4+ and promoted by NO3. Media for isolated root culture contain no NH4+, or very little. Although roots are able to take up nitrate ions from solutions, which become progressively more alkaline as assimilation proceeds, the same may not be true of cells, tissues and organs in vitro.

Shoot cultures. Media containing only nitrate nitrogen are used for the shoot culture of some plants, for example strawberry (Boxus, 1974), which can be cultured with 10.9 mM NO3 alone; supplementing the medium with 6 mM NH4+ causes phytotoxicity (Damiano, 1980). However, shoot cultures of strawberry grown without NH4+ can become chlorotic: adding a small amount of NH4NO3 (or another source of reduced nitrogen) to the medium at the last proliferation stage, or to the rooting medium, may then give more fully developed plants with green leaves (Zimmerman, 1981; Piagnani and Eccher, 1988). On some occasions it is necessary to eliminate or reduce NH4+ from the medium for shoot cultures to prevent hyperhydricity.

2.1.9. Nitrogen supply and morphogenesis

Morphogenesis is influenced by the total amount of nitrogen provided in the medium and, for most purposes, a supply of both reduced nitrogen and nitrate seems to be necessary. The requirement for both forms of nitrogen in a particular plant species can only be determined by a carefully controlled experiment: simply leaving out one component of a normal medium gives an incomplete picture. For example, cotyledons of lettuce failed to initiate buds when NH4NO3 was omitted from Miller (1961) salts and instead formed masses of callus (Doerschug and Miller, 1967): was this result due to the elimination of NH4+, or to reducing the total nitrogen content of the medium to one third of its original value?

Importance of the nitrate/ammonium balance. The importance of the relative proportions of NO3 and NH4+ has been demonstrated during indirect morphogenesis and the growth of regenerated plants. Grimes and Hodges (1990) found that although the initial cellular events which led to plant regeneration from embryo callus of indica rice, were supported in media in which total nitrogen ranged from 25 to 45 mM and the NO3 to NH4+ ratio varied from 50:50 to 85:15 (Table 3.5), differentiation and growth were affected by very small alterations to the NO3 to NH4+ ratio. Changing it from 80:20 (N6) medium, to 75:20, brought about a 3-fold increase in plant height and root growth, whereas lowering it below 75:25, resulted in short shoots with thick roots. Atypical growth, resulting from an unsuitable balance of nitrate and ammonium, has also been noted in other plants. It gave rise to abnormal leaves in Adiantum capillus-veneris (Pais and Casal, 1987); the absence of ammonium in the medium caused newly initiated roots of Carica papaya to be abnormally thickened, and to have few lateral branches (Drew, 1987). Shoot cultures may survive low temperature storage more effectively when maintained on a medium containing less NH4NO3 than in MS medium (Moriguchi and Yamaki, 1989).

By comparing different strengths of Heller (1953; 1955) and MS media, and varying the NH4NO3 and NaNO3 levels in both, David (1972) was led to the conclusion that the principal ingredient in MS favouring differentiation in Maritime pine explants is

NH4NO3. However, in embryonic expiants of Pinus strobus, adventitious shoot formation was better on Schenk and Hildebrandt (1972) medium than on MS

(which induced more callus formation). The difference in the ammonium level of the two media was mainly responsible (Flinn and Webb, 1986).

Table 3.4 Examples of adjustments of the nitrogen content of MS medium, which resulted in improved growth or morphogenesis. In each case, only NO3_ and NH4+ were changed, the rest of the medium being the same except for K+ and Cl_

Plant species

Type of culture

Results

(mM)

Ratio of NO3" to nh4+

Total N (mM)

Reference

Nicotiana tabacum

Callus

Optimum callus growth

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