CH (50 mg/l) (500 mg/l was inhibitory)
Gupta et al. (1987)
Chu and Hill (1988) MN6
Serine, proline, arinine, aspartic acid and alanine (each at 40 mg/l) + glutamine (400 mg/l)
Chu and Hill (1988)
CH (250 mg/l)
Mauro et al. (1986);
Zygotic embryo callus
Chu et al. (1975) N6
Proline (20-25 mM)
Kamo et al. (1985) Armstrong and Green (1985)
authors have pointed out that CH itself is also a source of phosphate. For example, Bridson (1978) and some chemical catalogues, show that some casein hydrolysates normally contain about 1.3g P2O5 per 100g. The addition of 2 g/l of CH will therefore increase the phosphate content of MS medium by 11% and that of White (1954) medium by 44% (assuming complete phosphate availability).
• A capacity to act as chelating agents. Some amino acids can act as chelating agents (see later in section on chelates)
• Enhanced nitrogen assimilation. Glutamine and glutamic acid are directly involved in the assimilation of NH4+. A direct supply of these amino acids should therefore enhance the utilization of both nitrate and ammonium nitrogen and its conversion into amino acids.
• A replacement for toxic ammonium ions.
Certain plant tissues are particularly sensitive to NH4+. Ochatt and Caso (1986) and Ochatt and Power (1988a, b) found that protoplasts of Pyrus spp. would not tolerate the ion, and that to obtain sustained cell division it was necessary to eliminate it from MS medium, and use 50 mg/l casein hydrolysate as a source of reduced nitrogen. CH can however be extremely toxic to freshly isolated protoplasts of some species and varieties of plants (Ranch and Widholm, 1980; Russell and McCown, 1988). Conifer tissues too are unable to cope with high concentrations of NH4+, but cultures can be supplied with equivalent levels of reduced nitrogen in the form of amino acids without the occurrence of toxicity (Durzan, 1982). In soybean suspension cultures, the high level of ammonium in MS medium has been shown to inhibit isocitrate dehydrogenase (a Krebs' cycle enzyme) and glutamine synthetase, which contribute to the conversion of NH4+ to glutamine (Gamborg and Shyluk, 1970). • Adjustment of intracellular pH. As intracellular pH is important for the activation of sea urchin eggs, and amino acids can promote embryogenesis, Nuti Ronchi et al., (1984) speculated that the uptake and assimilation of amino acids might help to regulate cellular pH in plants.
As mentioned before, there is commonly a minimum inoculation density below which growth cannot be initiated in vitro. This minimum varies according to both the source of the cells and the nature of the medium. It can usually be lowered by employing a 'conditioned' medium (i.e. a fresh medium into which the products of another medium in which cells are actively growing, have been added). Alternatively, initial growth at low densities can be supported by the close presence of other actively growing plant cells ('nurse cultures'). Compounds responsible for this effect must be freely diffusible from living cells and could include growth substances, reducing sugars, vitamins and amino acids. Addition of such supplements has been found to overcome the inhibited growth of some cells at low densities (Kao and Michayluk, 1975).
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