This term is used here as an alternative to plant hormones or phytohormones because it is still uncertain whether the chemical agents discussed can be strictly defined as hormones in the original sense of this term as taken from vertebrate physiology. At present such growth-regulating substances are commonly classified as auxins, cytokinins, and gibberellins although with the possible exception of the gibberellins it is not certain that these "classes" have each a uniform chemical basis.
They have been recognized primarily on the basis of biological tests the chemical specificity of which is in many cases very low and in other cases has not been critically examined. Furthermore, it may be antici
pated that further categories of growth-regulating substances will be recognized in plants as research progresses. This is emphasized by the recent isolation of the new growth-regulating substance, abscisic acid (previously described as "dormin" or as "abscisin I I " ) , which clearly differs in physiological and chemical properties from the other classes mentioned above (171, 557a).
As might be anticipated from the foregoing paragraph, our knowledge of the hormonal regulation of root growth and development is very un
satisfactory; the experimental basis of current hypothesis is extremely meager. It is true, however, that work with isolated root systems (in
cluding detopped plants) and with cultured roots has provided evidence that substances having the biological activity in certain tests of gib
berellins, cytokinins, and auxins are synthesized in roots.
Consideration of the role of such growth-regulating substances in tissue differentiation, lateral initiation, and cambial activity are con
sidered in subsequent sections. Here we are concerned with how far such substances are essential to cell division and cell expansion in the root and to assess what is known regarding their chemical nature. The auxins of roots have been under investigation for well over thirty years, and it is mainly in relation to auxin action in roots that root cultures have proved to be of some value. This aspect therefore forms the main theme of this section.
Roberts and Street (627) grouped excised roots on the basis of their behavior toward externally applied auxins into ( a ) those whose growth was either unaffected or inhibited by external auxin, for example, Lycopersicon esculentum (342, 745), and Acer rubrum ( 2 1 ) ; ( b ) those whose growth in culture was enhanced by an appropriate concentration of IAA or other auxin for example, Pinus sylvestris (677), Senecio vulgaris (152), Lupinus albus (219), Pisum sativum (75, 520), Zea mays (236, 366, 367), and Triticum vulgare (124, 148); ( c ) those whose
growth in culture depended upon an external supply of auxin, for example, Secale cereale (627). The evidence would also suggest that external auxin or a suitable auxin precursor may be essential for the growth of other cereal roots in culture; for the successful establishment of a clone of roots of the Tlilgendorf 6Γ variety of wheat by Ferguson (233) required the incorporation of either L- or D-tryptophan, or indol-3yl-acetic acid (IAA), or indol-3yl-acetonitrile (IAN), or other poten
tial precursors of indole auxin into the culture medium. Furthermore, although Roberts and Street (627), faced with the evidence then avail
able, were justified in concluding that the growth of some cultured roots was not enhanced by any external auxin, subsequent work has shown that a markedly enhanced growth of such roots can follow upon the addition of auxin to a medium of appropriately low sugar content.
These observations suggest that under any standard regime of culture the levels of natural auxin established may vary among the roots of differ
ent species, or between different strains within species, so that in some cases growth may be limited by auxin deficiency, whereas in others there may be a tendency for auxin to acumulate to inhibitory levels. For in
stance, Charles (152) showed that roots from different geographical strains of Senecio vulgaris differed markedly in their growth rates in a standard auxin-free medium, yet by appropriate applications of 2-naphth-oxyacetic acid (2-NOA) they could all be raised to a similar and very high level of growth. Furthermore the slow-growing strains required the higher concentrations of auxin, suggesting that their growth in the ab
sence of external auxin was determined by their different but in all cases, suboptimal levels of this natural growth regulator. In a number of other species (Lycopersicon esculentum, L. pimpinellifolium and Senecio vul
garis) a contrasted situation has been revealed for, with the progress of growth in culture, a natural auxin apparently accumulates and this first inhibits growth and later causes the cessation of activity of the root apical meristem. This phenomenon, which has been described as an
"aging" of the root meristems, is discussed in detail in a recent paper (734).
The fact that an external auxin may be essential to growth in culture, when viewed in the light of other experimental work with seedling roots and isolated root segments (16, 88, 412), must be regarded as strong evidence for the essentiality of auxin for root growth; so that roots obey Went's dictum "no auxin, no growth" (827).
Information concerning the factors which regulate auxin synthesis in roots is almost completely lacking and cannot expect to develop while the present confusion exists (see below) regarding the chemical identity of natural auxins. The marked interactions between sugar supply and
auxin response may indicate that auxin synthesis is markedly affected by carbohydrate level in the root cells. These interactions may, however, have a quite different basis, for various studies indicate that auxins can enhance the rate of sugar uptake by callus cultures (284-286, 288, 436, 856), that auxins direct the flow of sucrose to their site of application or accumulation (77), and that auxin and sucrose interact in the control of cambial initiation and vascular tissue differentiation (833). In line with such interpretations, Sutton, Scott, and Street (758) reported that concentrations of auxin strongly inhibitory to the linear growth of cul
tured wheat roots may actually increase the dry weight per culture, largely caused by wall thickening and starch accumulation so leading to increase in the percentage dry weight of the root tissues.
Some recent studies on the growth of excised wheat roots draw atten
tion to a further possible factor which may regulate the auxin level in cultured roots. In an auxin-free medium the linear growth, lateral initia
tion, and dry weight of these cultures is markedly enhanced by illumina
tion (737) (Fig. 17). A similar enhancement of growth can be achieved by supplying dark-grown cultures with either "autoclaved" tryptophan (25-50 mg/liter) or unheated tryptophan plus a low concentration of IAA (0.005 mg/liter) (148, 758). Furthermore, concentrations of "auto
claved" tryptophan which are stimulatory to "dark"-cultured roots ac
tually inhibit the growth of illuminated cultures. Such observations are clearly not incompatible with a light-activated synthesis in the root cells of some natural auxin and raise the question whether a photochemical reaction may be involved in the pathway of auxin synthesis in the whole plant. In this connection it may be recalled that illumination of cultured wheat roots enhances protein synthesis in the presence of similar reserves of soluble organic nitrogen to those present in dark-grown roots (200).
Since it is well known that marked increases in protein occur prior to cytokinesis and during the early stages of cell expansion (116, 354, 355, 701), it may be that the effect of light on protein synthesis is mediated through its effect on auxin synthesis or movement within the root.
Although under certain conditions auxin may be the "limiting factor"
controlling cell division and cell expansion in the root, there is a con
siderable body of evidence that auxin interacts with other growth-regulating substances in the control of these processes; that the balance between various natural growth regulators may be determinative in root growth and development. Thus it has been shown that lateral initiation and growth can be significantly influenced by applied gibberellic acid (Fig. 18), that gibberellic acid can speed up the "aging" of root meri
stems, presumably by acting synergistically with the natural root auxin, and that cultured roots contain natural gibberellins one of which seems
FIG. 17. Excised wheat roots (Alson, Elite 5 6 ) cultured in light for 1 4 days in White's medium containing 400 mg of casein hydrolyzate per liter. (1) Aerated with C 02 free air; ( 2 ) Aerated with air containing 5 % C 02. (Photographs by Beryl Talbot and Η. E. Street.)
to correspond very closely with gibberellin Ai (129, 130). By contrast kinetin acts as an "anti-aging" factor in prolonging the duration of ac
tivity of individual cultured root meristems and can antagonize the effect of externally applied gibberellic acid or 1-naphthaleneacetic acid ( N A A ) . There is now evidence for the synthesis of cytokinins in roots leading to export of these regulators to the shoot (147). Cytokinins are concentrated in seedling root apices (824) and can be detected in cul
tured tomato roots (unpublished results).
Control 1 0 gm/m l Gibberelli c aci d
FIG. 1 8 . Enhancement of lateral growth by addition of 0 . 0 1 mg of gibberellic acid per liter as illustrated by excised tomato roots cultured for 6 days in White's standard medium containing 2% sucrose (left) and in this medium containing gibberellic acid (right). (Shadowgraphs by D . N. Butcher.)
In studies with a number of cultured roots, including tomato, it is much easier to demonstrate stimulation of root growth from application of the synthetic auxins NAA and 2-naphthoxyacetic acid (2-NOA) than from application of IAA. These auxins, unlike IAA, will at higher con
centrations cause "aging" of root meristems under conditions which otherwise permit the indefinite culture of individual apices (734). More
over, antiauxins [a- (naphthylmethyl-sulfide) propionic acid and 1-naphthoxyacetic acid], which are effective in antagonizing "aging" under conditions of high sucrose supply, will also neutralize the inhibition of root growth which results from these auxins, but they are ineffective in reversing inhibition caused by externally applied IAA. Although one
cannot conclude from such observations that the natural auxin con-trolling cell division and the initiation of cell expansion in root tips is a naphthalene derivative (it might, however, be rewarding to search for natural compounds of this kind), they do raise the possibility that the auxin involved may not be IAA as such. Lahiri and Audus ( 4 0 3 , 4 0 4 ) have reported changes in auxin activity during germination at three sites
(designated, APi ? APH, and APm) on chromatograms of "acid ether-soluble" extracts of seedling roots of Vicia faba. One of these sites ( A P U ) was at an Rf which would not distinguish it from IAA: activity at this site only increased slowly and appeared to be principally located in the region of elongation. The region APm (Rf 0 . 8 - 1 . 0 in isobutanol/meth-anol/water) altered in activity with time in a way which suggests its synthesis and accumulation at the main apex meristem, and Lahiri and Audus suggested that this corresponded in its properties with the "aging"
auxin postulated from our studies with cultured roots. The findings of Lahiri and Audus have been confirmed and extended in our laboratory ( 7 3 6 ) . The zone AP^ after some purification ran as a single Ehrlich reactive spot when submitted to paper chromatography in isopropanol/
ammonia/water (Rf 0 . 8 3 ) , n-butanol/acetic acid/water (Rf 0 . 8 8 ) , 2 0 % K C 1 (Rf 0 . 7 5 ) and water (R, 0 . 7 5 ) . When submitted to low voltage paper electrophoresis at either pH 8 . 4 or pH 2 . 0 , it moved as a single spot toward the cathode. This purified APm not only was a growth promoter in the Avena straight growth coleoptile and Avena mesocotyl tests, but over a similar range of concentration promoted elongation growth in a cultured root test ( 8 5 9 ) .
The work quoted above raises the problem of the chemical nature and biological properties of root auxins, particularly of those which can be detected in cultured roots. The studies so far published on the auxins which can be detected in cultured and seedling roots serve only to emphasize the unsatisfactory state of our knowledge regarding these natural regulators ( 7 3 3 , 7 3 5 , 7 3 6 , 7 4 9 , 7 7 5 ) . The difficulty of what is being attempted in such studies should not be underestimated. It is difficult to obtain large quantities of young growing roots and there is evidence that the compounds being sought are present in the cells in very minute amounts and are very unstable. The use of large volumes of solvents alone poses the problem of introducing impurities in quantities which may heavily contaminate the concentrates containing auxin activity.
The purification techniques must be entirely empirical whilst the chemi-cal identity of the active compounds remains unresolved.
By utilizing the techniques of paper chromatography and electro-phoresis, thin-layer chromatography, and silica gel columns it has been possible to demonstrate that seedling and cultured roots contain a
number of substances which when substantially purified are active in the standard auxin bioassays, a number of these from their Ehrlich re
action and ultraviolet absorption spectra could be indoles, some of them react with ninhydrin indicative of amino acids, some initially ninhydrin-negative readily yield tryptophan (735). The active substances which have been most extensively purified are the "acidic ether-soluble" sub
stances; none of these are identical in properties with IAA and most can be distinguished from known natural indoles. A large fraction of the total auxin activity is preferentially water soluble but with the exception of tryptophan itself the active water-soluble auxins have not been puri
fied. Our experience coincides with that of Bennet-Clark, Younis, and Esnault (40) and of Burnett, Audus, and Zinsmeister (123); this is that whenever growth active zones inseparable from IAA by common chro
matographic solvents are examined by further techniques they can be shown to differ from this auxin. We would anticipate a similar fate for the zone reported as IAA on thin-layer chromatograms of methanol extracts of roots of Lens culinaris and Phaseolus vulgaris by Collet, Dubouchet, and Pilet (163). If this is correct we must conclude that IAA, if a normal constituent of roots, is present at levels which cannot be detected by existing techniques.
Further progress in understanding the auxin regulation of root growth must depend upon ascertaining whether the natural root auxins are indole compounds exclusively or in part and, if so, upon identifying them and studying their interconversions within cells. When this stage is reached it will still be necessary to identify which of these compounds have a direct growth-regulatory function. We are inclined to think of natural growth regulators as simple molecules; this thinking may be conditioned by the circumstance that we handle only compounds which can act as precursors of the active hormones of plant cells. If this is so, then after almost forty years of research in plant hormones, we are very far from understanding in chemical terms how they act to regulate plant growth.