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Morphogenesis influenced by total available nitrogen. Others have found that the total nitrogen content of culture media influences morphogenesis more than the relative ammonium concentration. Results of Margara and Leydecker (1978) indicated that adventitious shoot formation from rapeseed callus was optimal in media containing 30-45mM total nitrogen. The percentage of explants forming shoots was reduced on media containing smaller or greater amounts (e.g. on MS medium). Increasing the ratio of NH4+ to total N in media, from 0.20 to 0.33 was also detrimental. Similarly, Gertsson (1988) found that a small number of adventitious shoots was obtained on petiole segments of Senecio hybridus when the total nitrogen in MS medium was increased to 75 mM, but that an increased number of shoots was produced when the total nitrogen was reduced to

30 mM (while keeping the same ratio of NO3 to NH4+). Shoot production was more than doubled if, at the same time as the total N was reduced, the potassium ion concentration was fixed at 15 mM, instead of 20 mM.

The total amount of nitrogen in a medium was shown by Roest and Bokelmann (1975) to affect the number of adventitious shoots formed directly on Chrysanthemum pedicels. The combined amount of KNO3 + NH4NO3 in MS medium (60 mM), was adjusted as is shown in Fig. 3.3 while the ratio of NO3" to NH4+ (66:34) was unchanged. From 30-120 mM total nitrogen was optimal. However there was clearly a strong effect of genotype, because the cultivar 'Bravo' was much more sensitive to increased nitrogen than 'Super Yellow'.

Nitrogen x sugar interaction. The enhancement of morphogenesis caused by high nitrogen levels may not be apparent unless there is an adequate sucrose concentration in the medium (Margara and Rancillac, 1966; Gamborg et al., 1974). In Dendrobium, the uptake of NO3 is slower than that of NH4+. Uptake is dependent on the nature and concentration of the sugar in the medium, being slower in the presence of fructose than when sucrose or glucose are supplied (Hew et al., 1988). The rate of growth of Rosa 'Paul's Scarlet' suspensions was influenced by the ratio of NO3 to sucrose in the medium. A high ratio favoured the accumulation of reduced nitrogen, but not the most rapid rate of cell growth (Fletcher, 1980).

Fig. 3.3 The number of adventitious shoots formed directly on Chrysanthemum explants with increasing total nitrogen concentration in an otherwise normal MS medium [from data of Roest and Bokelmann, 1975]

Embryogenesis and embryo growth. It is 10 mM (from NH4Cl) in the presence of 12-40 mM

accepted that the presence of some reduced nitrogen is necessary for somatic embryogenesis in cell and callus cultures (Halperin and Wetherell, 1965; Reinert et al., 1967); but although reduced nitrogen compounds are beneficial to somatic embryo induction, apparently they are not essential until the stage of embryo development (Kamada and Harada, 1979). A relatively high level of both nitrate and ammonium ions then seems to be required. Some workers have also noted enhanced embryogenesis and/or improved embryo growth when media have been supplemented with amino acids in addition to NOs" and NH4+ Street (1979) thought that an optimum level of NH4+ for embryogenesis was about

[NO-T to NH4+ ratio, from 55:45 to 80:20; Total N 22-50 mM]

Walker and Sato (1981) obtained no embryogenesis in alfalfa callus in the absence of either ammonium or nitrate ions. Miller's medium [Miller (1961; 1963)] (12.5 mM NH4+) supported a high rate of embryogenesis:

[NO-~ to NH4+ ratio, 68:32; Total N 39.1 mM], but only a small number of embryos were produced on Schenk and Hildebrandt (1972) medium (SH): [NO-T to NH4+ ratio, 90:10, Total N 27.32 mM], unless it was supplemented with NH4+ from either ammonium carbamate, ammonium chloride or ammonium sulphate. An optimal level of ammonium in SH medium was 12.5 mM:

[NO3" to NH4+ ratio, then 66:34, total N, 37.22], although embryogenesis was still at a high level with 100 mM ammonium ion: [NO3" to NH4+ ratio, then, 20:80; total N, 124.72 mM].

By contrast, Coffea arabica leaf callus (Sondahl and Sharp, 1977), which was formed on a medium containing MS salts, was induced to become embryogenic by first being cultured on a medium with MS salts (and high auxin):

[NO-T to NH4+ ratio, 66:34; total N, 30.0 mM], and then moved to another medium containing MS salts with an extra 2850 mg/l KNO3: [NO3" to NH4+ ratio, 82:18; total N, 58.2 mM] (and low auxin)

Zygotic embryos. The presence of some reduced nitrogen in the growth medium is also required for the continued growth of zygotic embryos in culture. Nitrate alone is insufficient (Mauney et al., 1967; Norstog, 1967, 1973). An optimum concentration of NH4+ for the development of barley embryos in culture was 6.4 mM (Umbeck and Norstog, 1979). A similar provision seems to be necessary in most plants for the in vitro growth of somatic embryos.

Flower bud formation and growth. Nitrate was essential for the formation of adventitious buds on leaf segments of Begonia franconis. The greatest proportion of flower buds was obtained with 5 mM NO3" and 1.5 mM NH4+. Above this level, NH4+ promoted vegetative sprouts (Berghoef and Bruinsma, 1979b). The best in vitro growth of Begonia franconis flower buds detached from young inflorescences, occurred on a medium with 10-15 mM total nitrogen (NO3~ to NH4+ ratio, 50:50 to 67:33) (Berghoef and Bruinsma, 1979a). Detached flower buds of Cleome iberidella were found to grow best in vitro with 25 mM total nitrogen (NO3 to NH4+ ratio, 80:20) (De Jong and Bruinsma, 1974; De Jong et al., 1974), but the complete omission of NH4NO3 from MS medium, where the salts had been diluted to their original concentration, promoted the development (but not the initiation) of adventitious floral buds of Torenia fournieri (Tanimoto and Harada, 1979, 1981, 1982).

Effect on the action of growth regulants. The ratio of NO3 to NH4+ present in the culture medium has been found to affect the activity of plant growth substances and plant growth regulators. The mechanisms by which this occurs are not fully elucidated. It has been noted, for example, that cells will divide with less added cytokinin when the proportion of reduced nitrogen is reasonably high. To induce tobacco protoplasts to divide, it was necessary to add 0.5-2 mg/l BAP to a medium containing only NO3 nitrogen. The presence of glutamine or NH4+ in the medium together with NO3, reduced the cytokinin requirement, and division proceeded without any added cytokinin when urea, NH4+, or glutamine were the sole N-sources of the medium (Meyer and Abel, 1975b). Sargent and King (1974) found that soybean cells were dependent on cytokinin when cultured in a medium containing NO3 nitrogen, but independent of cytokinin when NH4+ was present as well.

The relative proportion of nitrate and ammonium ions also affects the response of cells to auxin growth regulators in terms of both cell division and morphogenesis. It is possible that this is through the control of intracellular pH (see below). Carrot cultures that produce somatic embryos when transferred from a high- to a low-auxin medium, can also be induced into embryogenesis in a high-auxin medium, if it contains adequate reduced nitrogen. Only root initials are formed in high-auxin media which do not contain reduced nitrogen (Halperin, 1967). The number of plants regenerated from rice callus grown on Chu et al., (1975) medium containing 0.5 mg/l 2,4-D, depended on the ratio of NO3 to NH4+. It was high in the unaltered medium (ratio 4:1), but considerably less if, with the same total N, the ratio of the two ions was changed to 1:1 (Grimes and Hodges, 1990).

Cells of Antirrhinum majus regenerated from isolated protoplasts were stimulated to divide with a reduced quantity of auxin in a medium containing 39.77 mM total nitrogen

by adding further ammonium ion to give a total nitrogen content of 54.72 mM

[NO-T to NH4+ ratio, 54:46 (1.19)]. or, alternatively, 400 mg/l of casein hydrolysate (Poirier-Hamon et al., 1974).

In experiments of Koetje et al., (1989) and Grimes and Hodges (1990) (Table 3.5), when the NO3 to NH4+ ratio in N6 medium was 80:20, there was a strong dose response curve to the auxin 2,4-D with 0.5 mg/l being the best concentration to induce embryogenesis in Oryza sativa callus; if the medium was modified, so that the NO3- to NH4+ ratio was 66:34 or 50:50, 2,4-D was less effective, and there was little difference in the number of plants regenerated between 0.5 and 3 mg/l 2,4-D. The ratio of NO3 to NH4+ therefore seemed either to to alter the sensitivity of cells to the auxin, or to affect its uptake or rate of metabolism.

Walker and Sato (1981) also found that the proportion of ammonium ion in the medium can influence the way in which growth regulants control morphogenesis. Having been placed for 3 days on a medium which would normally induce root formation [Schenk and Hildebrandt (1972) medium containing 5 mM 2,4-D and 50 mM kinetin], suspension cultured cells were subsequently plated on a modification of the same medium (which contains 24.8 mM NO3) without regulants, in which the concentration of NH4+ had been adjusted to various levels. Table 3.6 shows that the morphogenesis experienced, depended on the concentration of ammonia in the regeneration medium. Media containing high levels of ammonium ion would have tended to become acid, especially as the extra ammonium was added as ammonium sulphate. Possibly this affected the uptake or action of the regulants?

2.1.10. Addition of amino acids

Amino acids can be added to plant media to satisfy the requirement of cultures for reduced nitrogen, but as they are expensive to purchase, they will only be used in media for mass propagation where this results in improved results. For most tissue culture purposes, the addition of amino acids may be unnecessary, providing media contain adequate amounts, and correct proportions, of nitrate and ammonium ions. For example, Murashige and Skoog (1962) found that when cultures were grown on media such as Heller (1953; 1955), Nitsch and Nitsch (1956) N1, and Hildebrandt et al, (1946) Tobacco, containing sub-optimal amounts of inorganic chemicals, a casein hydrolysate (consisting mainly of a mixture of amino acids, see later) substantially increased the yield of tobacco callus, whereas it gave only marginal increases in yield when added to their revised MS medium. Arginine (0.287 mM) increased the growth of sugar cane callus and suspension cultures grown on Nickell and Maretzski (1969) medium (Nickell and Maretzski, 1969) but was without effect on cultures of this plant grown on a medium based on Scowcroft and Adamson (1976) CS5 macronutrients (Larkin, 1982).

It is noticeable from the literature that organic supplements (particularly amino acids) have been especially beneficial for growth or morphogenesis when cells or tissues were cultured on media such as White (1943a), which do not contain ammonium ions. White (1937) and Bonner and Addicott (1937), for example, used known amino acids to replace the variable mixture provided by yeast extract. For the culture of Picea glauca callus, Reinert and White (1956) supplemented Risser and White (1964) medium with 17 supplementary amino acids, and similar, or greater numbers, were used by Torrey and Reinert (1961) and Filner (1965) in White (1943) medium for the culture of carrot, Convolvulus arvensis, Haplopappus gracilis and tobacco tissues.

Dependence on the nitrate to ammonium ratio. Grimes and Hodges (1990) found that when both NO3 and NH4+ are present in the medium, the response to organic nitrogen depends on the ratio of these two ions. Twice as many plants were regenerated from embryogenic rice callus when 1g/l CH was added to Chu et al., (1975) N6 medium, providing the proportion of NO3 to NH4+ was also changed to 1 (50:50). There was little response to CH with the same amount of total N in the medium, if the NO3"/NH4+ ratio was 4 (i.e. 80%:20%, as in the original medium), or more.

2.1.11. Amino acids as the sole N source

As most of the inorganic nitrogen supplied in culture media is converted by plant tissues to amino acids, which are then assimilated into proteins, it should be possible to culture plants on media in which amino acids are the only nitrogen source. This has been demonstrated: for example, Nicotiana tabacum callus can be cultured on MS salts lacking NO3" and NH4+ (but with an extra 20.6 mM K+), if 0.1 mM glycine, 1mM arginine, 2 mM aspartic acid and 6 mM glutamine are added (Muller and Grafe, 1978); wild carrot suspensions can be grown on a medium

Table 3.6 The effect of total nitrogen, and the ratio of NO3 to NH4+ on the type of organ produced by alfalfa cells which have been subcultured on a root-inducing medium (Walker and Sato, 1981)

Type of organ

nh4+

no3"

Ratio of NO3" to

produced

(mM)

(mM)

nh4+

Roots

<2.5

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