Rchch2 Cooh

(where R = functional groups)

Unfortunately the particular amino acid, or mixture of amino acids, which promotes growth or morphogenesis in one species, may not do so in another. For instance, L-a-alanine, glutamine, asparagine, aspartic acid, glutamic acid, arginine and proline could serve as a source of reduced nitrogen in a medium containing 20 mM NO3 , and were effective in promoting embryogenesis in Daucus carota callus and suspensions, but lysine, valine, histidine, leucine and methionine were ineffective (Kamada and Harada, 1982).

Competitive inhibition. Some amino acids are growth inhibitory at fairly low concentrations and this is particularly observed when mixtures of two or more amino acids are added to media. Inhibition is thought to be due to the competitive interaction of one compound with another. In oat embryo cultures, phenylalanine and L-tyrosine antagonise each other, as do L-leucine and DL-valine; DL-isoleucine and DL-valine, and L-arginine and L-lysine (Harris, 1956). Lysine and threonine often exert a cooperative inhibition when present together, but do not inhibit growth when added to a medium singly (Cattoir-Reynaerts et al., 1981).

Glycine. Glycine is an ingredient of many media. It has usually been added in small amounts, and has been included by some workers amongst the vitamin ingredients. Despite frequent use, it is difficult to find hard evidence that glycine is really essential for so many tissue cultures, but possibly it helps to protect cell membranes from osmotic and temperature stress (Orczyk and Malepszy, 1985).

White (1939) showed that isolated tomato roots grew better when his medium was supplemented with glycine rather than yeast extract and that glycine could replace the mixture of nine amino acids that had been used earlier. It was employed as an organic component by Skoog (1944) and continued to be used in his laboratory until the experiments of Murashige and Skoog (1962). They adopted the kinds and amounts of organic growth factors specified by White (1943a) and so retained 2 mg/l glycine in their medium without further testing.

Linsmaier and Skoog (1965), furthering the study of medium components to organic ingredients, omitted glycine from MS medium and discovered that low concentrations of it had no visible effect on the growth of tobacco callus, while at 20 mg/l it depressed growth. No doubt the success of MS medium has caused the 2 mg/l glycine of Skoog (1944) to be copied in many subsequent experiments. Many workers overlook the later Linsmaier and Skoog paper.

Casein and other protein hydrolysates. Proteins, which have been hydrolysed by acid, or enzymes, and so broken down into smaller molecules, are less costly than identified amino acids. The degree of degradation varies: some protein hydrolysates consist of mixtures of amino acids together with other nitrogenous compounds such as peptide fragments, vitamins, and elements which might (if they can form inorganic ions, or are associated with organic compounds that can be taken up by plant tissues) be able to serve as macro- or micro-elements. Peptones are prepared from one or several proteins in a similar fashion but generally consist of low molecular weight proteins. Although protein hydrolysates are a convenient source of substances which may promote plant growth, they are by nature relatively undefined supplements. The proportion of individual amino acids in different hydrolysates depends on the nature of the source protein and the method by which the product has been prepared.

The hydrolysate most often used in culture media is that of the milk protein, casein, although lactalbumin hydrolysate has been employed (La Motte and Lersten, 1971). Peptones and tryptone have been used less frequently, but there are reports of their having been added to media with advantage (e.g. Muralidhar and Mehta, 1982; Pierik et al., 1988). Casein hydrolysates can be a source of calcium, phosphate, several microelements, vitamins and, most importantly, a mixture of up to 18 amino acids. Several casein hydrolysates (CH) are available commercially but their value for plant tissue culture can vary considerably. Acid hydrolysis can denature some amino acids and so products prepared by enzymatic hydolysis are to be preferred. The best can be excellent sources of reduced nitrogen, as they can contain a relatively large amount of glutamine.

Casein hydrolysate produces an improvement in the growth of Cardamine pratensis and Silene alba suspensions, only if the medium is deficient in phosphorus. Glutamine has the same effect; it is the most common amino acid in CH, and its synthesis requires ATP. For these reasons, Bister-Miel et al., (1985) concluded that CH overcomes the shortage of glutamine when there is insufficient phosphorus for adequate biosynthesis.

However several investigators have concluded that casein hydrolysate itself is more effective for plant culture than the addition of the major amino acids which it provides. This has led to speculation that CH might contain some unknown growth-promoting factor (Inoue and Maeda, 1982). In prepared mixtures of amino acids resembling those in CH, competitive inhibition between some of the constituents is often observed. For instance, the induction of embryogenesis in carrot cell suspensions on a medium containing glutamine as the only nitrogen source, was partly inhibited by the further addition of L-amino acids similar in composition to those in CH. This suppression was mainly caused by the L-tyrosine in the mixture (Anderson, 1976).

There may be a limit to the amount of CH, which can be safely added to a medium. Anstis and Northcote (1973) reported that the brand of CH known as 'N-Z-amine', can produce toxic substances if concentrated solutions are heated, or if solutions are frozen and thawed several times. Possibly these are reasons why mixtures of amino acids occasionally provide more valuable supplements than CH. Nicotiana tabacum callus grew better on a nitrogen-free MS medium when a mixture of the amino acids L-glutamine (6 mM), L-aspartic acid (2 mM), L-arginine (1 mM) and glycine (0.1 mM) was added, rather than 2 g/l casein hydrolysate (which would have provided about 2 mM glutamine, 0.6 mM aspartic acid, 0.2 mM arginine and 0.3 mM glycine) (Muller and Grafe, 1978).

2.1.13. Beneficial effects of amino acid additions

Improved growth. The growth rate of cell suspensions is frequently increased by the addition of casein hydrolysate or one or more amino acids (particularly glutamine) to media containing both nitrate and ammonium ions. Some workers have included a mixture of several amino acids in their medium without commenting on how they improved growth. In other cases the benefit resulting from a specific compound has been clearly shown. The lag phase of growth in suspensions of Pseudotsuga menziesii cultured on Cheng (1977; 1978) medium was eliminated by the addition of 50 mM glutamine, and the final dry weight of cells was 4 times that produced on the unammended medium (Kirby, 1982). Similarly the rate of growth of Actinidia chinensis suspensions was improved by the addition of 5mM glutamine (Suezawa et al., 1988) and those of Prunus amygdalus cv. 'Ferragnes' could not be maintained unless 0.2% casamino acids was added to the medium (Rugini and Verma, 1982). Molnar (1988b) found that the growth of Brassica nigra cell suspensions was improved by adding 1-4 g/l CH or a mixture of 4 mM alanine, 4 mM glutamine and 1 mM glutamic acid. In this case the medium contained MS salts (but less iron) and B5 vitamins.

Amino acid supplements have also been used to boost the rate of growth of callus cultures. For instance, Short and Torrey (1972) added 5 amino acids and urea to a medium containing MS salts for the culture of pea root callus, and Sandstedt and Skoog (1960) found that aspartic and glutamic acids promoted the growth of tobacco callus as much as a mixture of several amino acids (such as found in yeast extract). Glutamic acid seemed to be primarily responsible for the growth promotion of sweet clover callus caused by casein hydrolysate on a medium containing 26.6 mM NO3", 12.5 mM NH4+ and 2.0 mM PO43- (Taira et al., 1977).

Amino acids are often added to media for protoplast culture. It was essential to add 2 mM glutamine and 2 mM asparagine to a medium containing MS salts, to obtain cell division, colony growth and plantlet differentiation from Trigonella protoplasts (Shekhawat and Galston, 1983).

Shoot cultures. Many shoot cultures are grown on MS medium containing glycine, although in most cases the amino acid is probably not an essential ingredient. Usually it is unnecessary to add amino acids to media supporting shoot cultures, but methionine may represent a special case. Druart (1988) found that adding 50-100 mg/l L-methionine to the medium seemed to stimulate cytokinin activity and caused cultures of Prunus glandulosa var. sinensis to have high propagation rates through several subcultures. This promotive effect of L-methionine was thought to be due to it acting as a precursor of ethylene (see Chapter 7). Glutamine inhibited the growth of apical domes excised from Coleus blumei shoots (Smith, 1981) and 50-100 mg/l glutamic acid inhibited shoot growth, the formation of axillary buds and shoot proliferation in cultures of woody plants (Druart, loc. cit.).

L-Citrulline is an important intermediate in nitrogen metabolism in the genus Alnus. The addition of 1.66 mM (4.99 mM NH2) to WPM medium improved the growth of A. cordata and A. subcordata shoot cultures (Cremiere et al., 1987).

Contaminants grow more rapidly on media containing amino acids. Casein hydrolysate is therefore sometimes added to the media for Stage I shoot cultures so that infected explants can be rejected quickly (Schulze, 1988). The health of shoots grown from seedling shoot tips of Feijoa (Acca) sellowiana was improved when 500 mg/l CH was added to Boxus (1974) medium (which does not contain ammonium ions) (Bhojwani et al., 1987).

Organogenesis. The presence of amino acids can enhance morphogenesis, either when they provide the only source of reduced nitrogen, or when they are used as a supplement to a medium containing both NO3 and NH4+. In a medium containing 25 mM nitrate, but no NH4+, direct adventitious shoot formation on cauliflower peduncle explants was induced by the addition of a mixture of the amino acids asparagine, proline, tyrosine and phenylalanine, each at a concentration of only 0.1 mM. (Margara, 1969b). A high rate of adventitious shoot regeneration and embryogenesis, from Beta vulgaris petioles or petiole callus, was achieved on a medium comprised of several amino acids and a complex vitamin mixture with MS salts (Freytag et al., 1988). The addition of CH to MS medium was found to be essential for shoot formation from callus (Chand and Roy, 1981).

Adding only 1-10 mg/l of either L-leucine or L-isoleucine to Gamborg et al., (1968) B5 medium, decreased callus growth of Brassica oleracea var capitata, but increased adventitious shoot formation. Basu et al., (1989) thought that this might be due to these amino acids being negative effectors of threonine deaminase (TD) enzyme, the activity of which was diminished in their presence. Threonine, methionine and pyruvic acid, which increased callus growth in this species, enhanced TD activity.

There are several examples of the amino acid L-asparagine being able to stimulate morphogenesis. This may be because it too can be a precursor of ethylene (Durzan, 1982), the biosynthesis of which may be increased by greater substrate availability. Kamada and Harada (1977) found that the addition of 5 mM L-asparagine stimulated both callus and bud formation in stem segments of Torenia fournieri, while alanine (and, to a lesser extent, glutamic acid) increased flower bud formation from Torenia internode segments when both an auxin and a cytokinin were present. An increase in the number of adventitious buds formed on the cotyledons and hypocotyl of Chamaecyparis obtusa seedlings occurred when 1.37 mM glutamine and 1.51 mM asparagine were added together to Campbell and Durzan (1975) medium, but not when they were supplied on their own (Ishii, 1986). L-asparagine was also added to MS medium by Green and Phillips (1975), to obtain plant regeneration from tissue cultures of maize; adding it to Finer and Nagasawa (1988) 10A4ON medium caused there to be more embryogenic clumps in Glycine max suspension cultures (Finer and Nagasawa, 1988).

Amino acid additions do not invariably enhance morphogenesis. Supplementing Linsmaier and Skoog (1965) medium with 0.5-5 mM glutamine, caused callus of Zamia latifolia to show greatly decreased organogenesis (Webb and Rivera, 1981) and ammending Linsmaier and Skoog (1965) medium with 100 mg/l CH, prevented adventitious shoot formation from stem internode callus of apple and cherry rootstocks (James et al., 1984).

Embryogenesis. The presence the ammonium ion is usually sufficient for the induction of embryogenesis in callus or suspension cultures containing NO3, but on media where NH4+ is lacking [e.g. White (1954)], casein hydrolysate, or an amino acid such as alanine, or glutamine, is often promotory (Ranga Swamy, 1958; Ammirato and Steward, 1971; Street, 1979). For embryogenesis in carrot cultures, Wetherell and Dougall (1976) have shown that in a medium containing potassium nitrate, reduced nitrogen in the form of ammonium chloride matched the effectiveness of an equivalent concentration of nitrogen from casein hydrolysate. Casein hydrolysate could be replaced by glutamine, glutamic acid, urea or alanine. Suspensions of wild carrot cells grew and produced somatic embryos on a medium containing either glutamine or CH as the sole nitrogen source (Anderson, 1976).

There have also been many reports of embryogenesis being promoted by the addition of casein hydrolysate, or one or more specific amino acids, when both NO3 and NH4+ were available in the medium. Some examples are given in Table 3.7. In many cases, embryogenic callus and/or embryo formation did not occur without the presence of the amino acid source, suggesting that without amino acid, the medium was deficient in NH4+ or total nitrogen. Armstrong and Green (1985) found that the frequency of friable callus and somatic embryo formation from immature embryos of Zea mays increased almost linearly with the addition of up to 25 mM proline to Chu et al., (1975) N6 medium

[total N, 34.99 mM; NO3/NH4 ratio, 3.99], but there was no benefit from adding proline to MS medium (containing 150 mg/l asparagine hydrate).

The growth of somatic embryos can also be affected by the availability of reduced nitrogen. That of Coronilla varia embryos was poor on Gamborg et al., (1968) B5 medium (Total N 26.74 mM; NH4+ 2.02 mM), unless 10 mM asparagine or 20 mM NH4Cl was added to the medium, or unless the embryos were moved to Saunders and Bingham (1972) BOi2Y medium, which has 37.81 mM total inorganic N, 12.49 mM NH4+ and 2000 mg/l casein hydrolysate (approx. 9.9 mM NH4+ equivalence) (Moyer and Gustine, 1984). However, the germination of Triticum aestivum somatic embryos was completely prevented by adding 800 mg/l CH to MS medium (Ozias Akins and Vasil, 1982; Carman et al., 1988).

Culture of immature cotyledons. Young storage cotyledons isolated from immature zygotic embryos accumulate protein efficiently when cultured with amino acids in a medium without nitrate and ammonium ions. A medium such as that of Millerd et al. (1975), or of Thompson et al., (1977) is normally used, but where the effect of different amino acids on protein assimilation is being studied, the amino acid content of the medium is varied. Glutamine is often found to be the most efficient nitrogen source for this purpose (Thompson et al., 1977; Haga and Sodek, 1987), but protein increase from culture with asparagine and glutamate (glutamic acid) is usually also significant (Lea et al., 1979).

2.1.14. Causes of the stimulatory effect of amino acids

We may conclude therefore, that for many cultural purposes, amino acids are not essential media components; but their addition as identified pure compounds, or more cheaply through casein hydrolysates, can be an easy way of ensuring against medium deficiency, or of providing a source of nitrogen that is immediately available to cultured cells or tissues. An observation by Murashige and Skoog (1962) that the presence of casein hydrolysate allowed vigorous organ development over a broader range of IAA and kinetin levels, may be of significance.

In gram moles per litre, amino acids can be a much more efficient source of reduced nitrogen than ammonium compounds. For instance, the mixture of amino acids provided by 400 mg/l of casein hydrolysate (containing at most as much reduced nitrogen as 3.3 mM NH4+) was as effective as 14.95 mM NH4Cl in stimulating the division of protoplast-derived cells of Antirrhinum (Poirier-Hamon et al., 1974).

Why should this be, and why can additions of amino acids (sometimes in comparatively small amounts) stimulate growth or morphogenesis when added to media, which already contain large amounts of NH4+? Some hypotheses, which have been advanced are:

• Conservation of ATP - alleviating phosphate deficiency. Durzan (1982) pointed out that when plant tissues take up the ammonium ion, they consume adenosine tri-phosphate (ATP) in converting it to amino acids. If suitable amino acids are available from the medium, some of ATP may be conserved. Bister-Miel et al., (1985) noted that CH promoted growth in cultures where phosphate became growth-limiting. They suggested that amino acids compensated for phosphate deficiency. With the plant well supplied with amino acids, some of the phosphate, which is normally used for ATP production can be diverted to other uses. Several

Table 3.7 Some examples of the promotion of embryogenesis by amino acids in media containing NO3- and NH4

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