The inorganic salt solutions now used to supply essential mineral ele-ments to plant tissue cultures have been developed by modifying the solutions used by Gautheret (255, 256) and by White (849) in their pioneer work on the conditions necessary for the continuous growth in culture of tissues of callus origin (738). A number of workers demon-strated that the inorganic salt additions of White and Gautheret were suboptimal (121, 315, 336, 516); in particular enhanced growth of a number of tissues was obtained by using considerably enhanced levels of potassium and nitrogen and some increase in the level of phosphorus (121, 516, 547, 863). Consideration of the extent to which these enhanced levels of potassium, nitrogen, and phosphorus promote growth through activating certain biosynthetic potentialities of the cultured tissue will be deferred to a later section of this chapter, where attention is directed to differences in physiology between normal, habituated, and crown gall tissue cultures of the same plant origin.2
2 In this area the same problems that have beset those who interpret mineral nutrition of rooted plants also arise. Among these are the distinctions, not always
Determination of which mineral elements are essential for callus cultures and descriptions of the effects of deficiencies of these elements have been undertaken by Heller and his associates. Heller (315) worked with tissues capable of active growth on a defined medium and dispensed with the use of agar (which is a source of calcium, magnesium, and a number of microelements). The cultures were supported at the surface of the liquid culture medium by ashless filter paper (Fig. 34, p. 118).
Growth was enhanced if good contact was ensured by interposing, be
tween the tissue and the paper, a film of silica gel. A modification of this technique, which permits of intermittent renewal of the culture medium, still further enhances growth, apparently mainly by renewing the gas phase in the culture tubes (317).
Using this liquid medium technique, Heller has demonstrated (not unexpectedly) that callus cultures (like whole plants) require nitrogen, potassium, calcium, magnesium, phosphorus, sulfur, iron, zinc, boron, manganese, and copper. The question of requirements of chloride and iodide does not seem to have been examined critically. Sodium only slightly delays the death of tissues deprived of potassium. The other established requirements were similarly shown to be specific.
As in the case of cultured roots, sulfur can be effectively supplied not only as sulfate but alternatively as sulfite, sulfide, cysteine, gluta
thione, or methionine (316) (see p. 15 et seq.). It is claimed that phos
phorus can be suplied not only as inorganic orthophosphate but as vari
ous sugar phosphates, although the possibility does not seem to have been excluded that such organic phosphates may suffer degradation in the external medium (607).
Riker and Gutsche (615), Nickell and Burkholder (536), and Heller (315) have reported that nitrogen is required as nitrate and that their callus cultures cannot, even at neutral pH, utilize nitrite or ammonium as effective sole sources of nitrogen. In view of recent studies on the utilization of ammonium by cultured roots (p. 23 et seq.), this conclusion should probably be submitted to more critical study. Shantz and Steward clear, between effects due to total supply and to concentration of a solute in question and the way absorption may be affected by aeration of the medium, renewal of the solution at the absorbing surface, as well as by the metabolic activity of the absorbing cells. By their innate ability to accumulate ions (cf. Chap
ter 4, Volume II), cells, and roots, are often able to absorb them from great dilutions; nevertheless the access of ions to growing tissue may be much affected by the relative proportions of tissue to nutrient medium, and whether the tissue is grown in liquid, agitated or otherwise, or stationary on agar proliferating in air.
Lacking analyses of the initial tissue and of the tissue as grown, it is not always easy to distinguish, unequivocally, which has occurred or by what factors it may have been regulated. (Ed.).
( 6 6 0 ) found that urea could act in lieu of the most eifective combination of nitrate and amino acids for the growth of explanted carrot tissue.
Steinhart, Standifer, and Skoog ( 6 9 7 ) found that urea was an efficient source of nitrogen for spruce (Picea) tissue. The possibility that the activity of urea as a nitrogen source does not necessarily involve its con
version to carbon dioxide and ammonium has been raised ( 6 6 0 ) . Riker and Gutsche ( 6 1 5 ) found that the following single amino acids, although inferior to nitrate, could support the growth of sunflower (Helianthus) normal and crown gall tissues: alanine, glycine, arginine, glutamic acid, aspartic acid, and asparagine. Nitsch and Nitsch ( 5 4 8 ) reported that callus cultures of Helianthus tuherosus grew as well with alanine, γ-aminobutyric acid, glutamic acid, aspartic acid, glutamine, or urea as with nitrate during a 21-day growth period; and Demetriades ( 1 9 9 ) found for several normal callus cultures that aspartic acid and some
times glutamic acid or arginine could effectively replace the nitrate requirements. It must, however, be emphasized that for many cultured tissues single amino acid or amino acid mixtures are either completely ineffective as sole sources of nitrogen or are markedly inferior to nitrate.
Any seeming ineffectiveness of amino acids cannot be due to inability of the cultured tissues to absorb amino acids, for when supplied as
1 4C-labeled compounds they are readily absorbed and metabolized. The results suggest rather that the absorbed amino acids may not always be actively deaminated or that their deamination does not yield am
monium at sites where it can be effectively used for the synthesis of such primary products of nitrate assimilation as glutamic acid. An alter
native explanation, particularly where amino acid mixtures containing key amino acids like glutamic acid, aspartic acid, alanine, and arginine are ineffective, is that the cells have an absolute requirement for nitrate (note the evidence quoted above for the ineffectiveness of ammonium) because certain intermediates in nitrate reduction are involved in the biosynthesis of other essential nitrogenous constituents. Balanced amino acid mixtures ( 3 0 8 , 6 3 9 ) , or particularly individual amino acids, them
selves ineffective as sole sources of nitrogen, may be significantly or quite dramatically stimulatory to growth in presence of nitrate ( 5 2 6 ,
5 4 8 , 5 7 4 ) .
It is a general experience with cultured carrot tissue that it responds to casein hydrolyzate, even in the presence of nitrate, if the growth factors present stimulate growth to its maximum (Chapter 8 ) . It has been argued, in such cases, that growth with nitrate alone is limited either by the level of particular amino acids or by the rate of synthesis of amino acids, like glutamic acid, which occupy a central role in the synthesis of other amino acids and of other nitrogenous cell
consti-tuents. Clearly, to test such hypotheses it is necessary to combine ob
servations on growth with analysis of the tissues and the use of iso-topically labeled compounds to test the biosynthetic potentialities of cultured versus normal tissue, a subject which is dealt with in Chap
ter 7. This is especially true when considering the nitrogen nutrition of plant tissue cultures, and in the attempt to assess how far their par
ticular nitrogen metabolism is a consequence of in vitro culture, i.e., of the transition from the quiescent state to actively growing cells
(Chapter 4, Vol. IVA). Papers there cited, going back even to the metab
olism of aerated potato slices have constantly stressed that the quiescent state of storage organs (potato tuber, carrot root, artichoke [Helianthus tuberosus] tuber, etc.) is often characterized by a high content of non
protein (amino acid and amide) nitrogen which may represent one-half to two-thirds of the total. The early events of growth induction (cf.
Chapter 4, Vol. IVA) commonly reduce this to much lower levels and, as the cells grow and synthesize protein, they retain a qualitatively different composition of nitrogen compounds in which asparagine and arginine are often less conspicuous whereas glutamine, alanine, and often y-aminobutyric acid persist. These considerations are also to be discussed later (Chapter 8 ) .
Menoret and Morel (482) also concluded, from studies with carrot root explants, that the conditions of tissue culture caused profound changes in their free amino acid composition. The tissue at the time of excision from the carrot root was rich in arginine, glutamine, aspara
gine, and glutamic acid. After a period in culture the tissue did not con
tain a detectable level of arginine, the amounts of the amides and of glutamic acid had markedly decreased relative to other amino acids and the alanine content had risen to account for 4 0 % of the free amino nitrogen of the tissue. Duranton (225) found that the tuber tissue of Helianthus tuberosus was extremely rich in arginine (300 mg per 100 gm fresh weight) but that the amino acid composition changed very rapidly when the tissue was excised and placed on culture medium. Arginine disappeared and the levels, particularly of proline but also of hydroxy-L-proline, glutamic acid, the amides, and alanine, also increased. This utilization of the store of arginine by tissue initiating growth in culture follows a different course than that of arginine utilization in the develop
ing shoot or seedling, where proline does not accumulate and its carbon becomes incorporated in newly synthesized organic acids, sugar, and chlorophyll pigments (226-228). Again arginine metabolism follows a still different course in crown gall tumor tissue of the same species, giving rise to various monosubstituted guanidines (see page 175). If amino acid metabolism is subjected to such marked modifications as a
consequence of growth in culture, further study of these changes and of the changing activities of the enzymes involved may represent a promising experimental approach to the investigation of differentiation and of the control mechanisms involved in cellular metabolism (cf.
Chapter 7 ) . Some reservations must, however, be made here, for Morel ( 5 0 1 ) found with normal tissues of Helianthus tuberosus that if kinetin was added to the culture medium used by Duranton (which contained auxin as the only growth factor added), then not only was growth enhanced but the metabolism of arginine followed much more closely the pattern observed during seedling growth and neither proline nor hydroxyproline accumulated.
Heller and Richez ( 3 1 8 ) and Devillers-Anson ( 2 1 1 ) in their studies on the iron nutrition of callus cultures have drawn attention to the influence of released metabolites on iron availability. It has been stressed in a previous section that to meet the iron requirement of roots cultured in media of pH above 5 . 2 it is necessary to add an effective chelating agent such as ethylenediaminetetraacetic acid ( E D T A ) (see Section II, B ) . This is not the case for the callus cultures studied by Heller, which released unidentified substances capable of chelating iron. Addi-tion of "spent" medium which had supported the growth of such callus cultures effectively substituted for EDTA in maintaining iron availability to cultured tomato roots at pH values of 6 . 0 or above.
This brief summary of some of the more interesting observations made on the inorganic and nitrogen nutrition of callus cultures indicates that their use has not hitherto made distinctive contributions to our under-standing of the involvement of mineral elements and simple organic nitrogen compounds in metabolism or of the mechanisms of solute up-take by plant cells.