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J. Cell Sci. 12, 37-53 (1973) Printed in Great Britain 37 DIFFERENCES IN PLOIDY AND DEGREE OF INTERCELLULAR CONTACT IN DIFFERENTIATING AND NONDIFFERENTIATING SYCAMORE CALLUSES K. WRIGHT AND D. H. NORTHCOTE Department of Biochemistry, University of Cambridge, Cambridge CB2 iQW, England SUMMARY Comparisons have been made between a sycamore callus (S4) isolated in 1970 and one first isolated in 1958 (S2). Incubation of S4 on media containing different concentrations of auxin and kinetin showed that the ratio of the hormones was the factor which controlled the growth pattern within the tissue. High ratios of auxin: cytokinin favoured a white friable tissue, whereas a compact callus with a much harder texture was formed at low ratios. Roots were differentiated at intermediate values. Callus (S2) has not been induced to undergo cellular differentiation. No soluble factors which were capable of stimulating differentiation in S2 were produced during the differentiation of S4, and S2 did not affect the ability of S4 to form roots. Divisions seen in suspension cultures of S4 callus were mainly diploid; the cells of S2 were predominantly tetraploid. When S2 suspensions were grown in the cytokinin-free medium used for S4 a considerable number of diploid cells were seen. A number of S2 clones have been isolated and all these developed chloroplasts on exposure to continuous light and, when grown on the medium used to induce differentiation in S4, five of them showed a growth pattern in which the cells were more closely packed than is usually the case in S2 callus. A greater degree of cell contact has also been induced by including polyethylene glycol in the medium and so restricting the availability of water to the callus. Under these conditions the cells must have a greater capacity for interaction with one another. The effect of tissue texture on the extent of cell contact and adhesion between the cells is discussed in relation to the composition of the plant cell wall and ideas on the totipotency of plant tissue cultures. INTRODUCTION Many callus tissues have been found to have the ability to undergo cellular differentiation. In some cases this occurs spontaneously, whereas in others the differentiation can be experimentally induced, usually by an appropriate supply of auxins and cytokinins. In this paper we examine some of the factors which control the differentiation and organization of sycamore callus. Cytokinins are known to exert an effect on cell division (Das, Patau & Skoog, 1956; Miller, Skoog, Von Saltza & Strong, 1958; Roberts, 1969) and differentiation is usually considered to be possible only immediately following cell division (Sinnott & Bloch, 1945; Fosket, 1968). In some instances, however, differentiation can be induced in parenchyma cells without immediate prior cytokinesis (Sachs, 1969). Fosket & Torrey (1969) have used soybean tissue to show that kinetin above a certain concentra- 38 K. Wright and D. H. Northcote tion is required for xylem element formation to occur, but that this is not a consequence of its effect on cell division. Kinetin can also affect the ploidy of a tissue (Torrey, 1959, 1961, 1967) and this is presumably connected with its role in cell division. Torrey (1959, 1961, 1967) found that when kinetin or yeast extract was present in the medium on which a pea-root callus was initiated the callus became tetraploid and lost its ability to differentiate roots. When kinetin was omitted from the medium however, the callus remained diploid and was still capable of organ formation. The degree of polyploidy of particular tissues within an intact plant is characteristic both of the tissue and of the plant (D'Amato, 1952) and the role of polyploidy as a factor in plant development has been reviewed by D'Amato (1964). A sycamore callus (S2) has not been induced to undergo cellular differentiation since its isolation in 1958. This tissue has been cultured on a medium containing coconutmilk, a component of which has been shown to have cytokinin activity (Zwar & Bruce, 1970). We have reported the induction of root formation from a new sycamore callus (S4) grown on a defined medium (Wright & Northcote, 1972) and the stimulus for this differentiation was the supply of kinetin in the growth medium. In the present investigations we examine the role of kinetin in the growth and differentiation of sycamore tissue cultures. A great many theoretical and practical consequences of tissue culture studies depend upon the ideas of totipotency and the ability of the cells to differentiate. It would be of great value, therefore, if a general method were available for the induction of differentiation within a callus. With this in mind we have examined some differences between the S2 and S4 tissues and describe some experiments in which we have induced each of the cultures to assume some of the properties of the other. MATERIALS AND METHODS Tissue culture The culture and isolation, of S4 callus have been described by Wright & Northcote (1972). The method of isolation and culture of S2 callus has been described by Stoddart & Northcote (1967) and except where stated it was grown on a medium containing coconut-milk, sucrose, 2,4-dichlorophenoxyacetic acid (2,4-D) and the same mineral salts and vitamins as were used by Heller (1953) (Table 1). S4 was grown on a medium based on Gamborg's (1966) PRL4 medium (Wright & Northcote, 1972) whose composition is shown in Table 1. Two per cent sucrose was supplied except where otherwise stated. All callus cultures were maintained in the dark at 26 ± 2 °C. Suspensions of cells in liquid media were grown in conicalflasksin an orbital shaker operating at 97 rev/min and maintained in the dark at 26 ± 1 °C. Suspension cultures (100 ml) in a 500-ml conical flask were subcultured every week by transferring 10 ml into 90 ml of fresh medium; sterile precautions were observed throughout (Wright & Northcote, 1972). Microscopy The techniques used have been described by Jones & Northcote (1972) and Wright & Northcote (1972). Differences between sycamore calluses 39 Table 1. Composition of media used for sycamore callus Medium for S2 Inorganic components, mg/1. NaH,PO 4 .H 2 O Na2HPO4 KC1 NaNO3 MgSO 4 .7H,O (NH4)2SO4 KNO 3 CaCl2.6H2O FeCl3 Fe2+EDTA ZnSO 4 .7H,O H3BO3 MnSO 4 .4H 2 O AICI3 NiCl 2 .6H 2 O KI CuSO 4 .5H 2 O Na 2 MoO 4 .2H,O CoCl 2 .6H 2 O Organic components, mg/1. w-Inositol Pyridoxine HC1 Nicotinic acid Calcium D-pantothenate Thiamine 2,4-Dichlorophenoxyacetic acid i-Naphthylacetic acid N-Z-amine type 'A', g/1. (Sheffield Chemical, N.Y., U.S.A Sucrose, g/1. Coconut milk, ml/1. 125 — 75° 600 250 — — 75 1 — 1 1 o-oi 0-03 0-03 o-oi 0-03 •— — Medium (PRL4) forS4 90 30 300 250 200 1000 220 — 28 3 3 13 — — o-75 025 0-25 0-25 — — — 100 I — I 10 — 6 — — 1 1 1 2 20 20 200 — Chromosome counting After 3 days a sample of the subculture was placed on a slide, excess moisture was removed, and the tissue was flooded with aceto-orcein and squashed between slide and coverslip (Roberts & Northcote, 1970). The preparations were examined using a Zeiss Ultraphot model II and the chromosomes were counted in squashes of 80-100 dividing cells for each culture. Occasional squashes were impossible to count and if cells of a particular ploidy level did not squash so well as the others the results will be unavoidably biased against these. Cloning procedure Samples were removed from liquid suspension cultures and plated thinly on to solidified medium in a Petri dish. The positions of single cells were noted with the aid of a dissecting microscope. After 5 weeks clones arising from these single cells were removed and grown alongside nurse tissue (Muir, Hildebrandt & Riker, 1954) on solidified medium contained in sterile plastic jars (Wright & Northcote, 1972) until the colony was large enough to be cultured alone. K. Wright and D. H. Northcote 4° 005 0-2 W NAA, mg/l. 0-5 W w W 0 01 20 08 10 10 W w W w W W 50 0-1 80 100 100 w 005 20 w 01 05 0-5 1 0 ;2 10 Wxp 02 20 0-25 W 0-4 05 1 25 0 25 0 63 0-125 Cxp 08 0063 Cxp 20 0025 Cxp Cxp 0-1 0-25 Cxp 0-4 Fig. i. The effect of different NAA and kinetin concentrations on the growth and differentiation of S4 callus. The results were recorded 8 weeks after subculture. # , Death; ^ , root formation; texture of the callus, W = white; C = compact (see text). Calluses in which xylem and phloem were found are marked (xp). The value in the bottom right-hand corner of each square is the ratio NAA:kinetin (w/w) in the incubation. RESULTS The effect of kinetin at different l-naphthylacetic acid (NAA) concentrations Kinetin has been identified as a factor necessary for differentiation to occur in S4 callus (Wright & Northcote, 1972). In order to investigate this further S4 callus was grown on media containing kinetin at concentrations of between o and 2 mg/1. and for each medium NAA was included over the concentration range 0-10 mg/1. (Fig. 1). The results after 8 weeks are shown in Fig. 1. Each experiment was performed at least in duplicate. The numbers in the bottom right-hand corner of each square show the ratios of NAA: kinetin (w/w) in the incubation. Calluses are classified as ' white' or 'compact'. Apart from their colour, white calluses were characterized by extensive growth, little if any differentiation and a much greater friability than the compact calluses which were very hard, had a red-brown pigmentation and usually contained Differences between sycamore calluses 41 some differentiated tissue that could be identified microscopically. The difference in the external appearances of these 2 types of tissue is illustrated in Fig. 3. The results summarized in Fig. 1 show that white calluses were produced on high NAA: kinetin ratios while low ratios resulted in compact tissues. At ratios varying between 0-5 and 20 the calluses had some external features of both the white and the compact tissues and roots often grew from the surface. Selected calluses grown on media containing extreme ratios of hormones were processed for microscopical examination and those incubations in which xylem and phloem were identified are appropriately marked in Fig. 1. Since it is difficult to be certain about negative results these are not marked on the diagram. It can be seen that xylem and phloem were formed even at the lowest NAA: kinetin ratios tested and some well developed areas were observed (Figs. 6 and 10). However, although it was qualitatively the same, differentiation seen in sections of tissues grown in the presence of these low hormone ratios was less extensive than that found in calluses where roots were formed (Wright & Northcote, 1972). Differentiation was found in only one case where the NAA concentration was 10 mg/1. and we have previously shown that differentiation did not occur in the absence of kinetin (Wright & Northcote, 1972). Kinetin has a striking effect on the size of the cells as well as on the extent of differentiation in the tissue. Figs. 9 and 10 show cross-sections through the surface areas of white and compact calluses. With a low NAA: kinetin ratio the cells were much smaller than in the absence of kinetin and there were localized areas of high mitotic activity. When no kinetin was supplied the cells were larger and much more uniform in size than those grown in the presence of kinetin. Some S4 callus has been subcultured on a kinetin-containing medium (PRL4 with 1 mg/1. NAA and o-i mg/1. kinetin) throughout 6 subcultures (approximately 12 months). No roots have been formed since the second subculture and sections of the callus show that only a few isolated areas contain differentiated tissue. The callus still retains its hard texture. Interaction between S2 and 1S4 calluses on kinetin-containing media From the above experiment and also from previous work (Wright & Northcote, 1972) a medium based on PRL4, that contains 1 mg/1. NAA and 2 % sucrose together with o-i mg/1. kinetin was shown to be suitable for testing the ability of sycamore callus to differentiate. Simpkins, Collin & Street (1970) did not report any differentiation when kinetin was added to the medium in which suspensions of the original isolate (S2) were growing. Tissues (S2 and S4) were cultured together on the medium described above in order to test whether any extracellular water-soluble substances produced by one tissue could affect the other. This technique is analogous to the' nurse tissue' procedure of Muir et al. (1954). Pieces of one tissue were arranged around one piece of the other on solidified medium in a 500-ml conical flask. In controls, tissue from only one callus was used and inocula came from the medium on which these calluses are normally maintained in this laboratory. The experimental arrangement is shown in Fig. 4. Roots were frequently produced by S4 when it surrounded S2, although no differentiation was found in S2. Callus S4 was still able to differentiate 42 K. Wright and D. H. Northcote when surrounded by S2 and controls containing only S4 formed xylem, phloem and roots while those containing only S2 did not. A sample of S2 from theflaskin which this was surrounded by S4 callus was removed to a further flask and surrounded by fresh S4 inocula. Again no differentiation was found in the S2 callus after growth had occurred and this result was also obtained when the S2 was subcultured for a third time. Finally, a sample of S2 from the third subculture was transferred to a medium containing all the nutrients and hormones described above and to which had been added a homogenate of approximately 20 g fresh weight of S4 callus which had been incubated for 3 weeks on a medium causing differentiation. Thus the aqueous factors present in differentiating S4 callus were made available to S2 tissue. After a lag period of 2 weeks the callus began to grow very rapidly but after growth for a further 4 weeks, sections of the callus revealed no differentiation within the S2 callus. The effect of kinetin on the ploidy of the calluses Roberts & Northcote (1970) established that S2 callus grown in suspension is predominantly tetraploid and their method of chromosome counting has been employed to study the chromosome number of S2 and S4 calluses grown on different media. After squashing and staining with aceto-orcein chromosome counts were made on between 80 and 100 dividing cells from suspension cultures. The chromosome number distribution of S2 tissue grown on its usual medium is shown in Fig. 2 A. The diploid number of sycamore is 52 (Foster, 1933) and while it is difficult to specify the accuracy of counts with high numbers of small chromosomes, the clustering about the tetraploid number is unmistakable. The corresponding distribution for S4 grown as a suspension in PRL4 with 1 mg/1. NAA is shown in Fig. 2 D and here the chromosome count is distributed about the diploid number although occasional tetraploid mitoses were seen. Thus there was a fundamental difference between the sycamore callus prepared in 1958 and the one isolated in May 1970. A series of experiments was carried out in order to grow S2 on a medium containing no kinetin and to facilitate future comparative studies PRL4 with 1 mg/1. NAA was chosen. A suspension in medium containing coconut milk was subcultured into one based on PRL4 that included 1 mg/1. NAA and o-i mg/1. kinetin; it was then transferred to a medium with a lower kinetin concentration (o-oi mg/1.) and finally into one with 1 mg/1. NAA but no kinetin. The chromosome number distributions of S2 that had been grown in PRL4 with 1 mg/1. NAA after 5 and 8 subcultures are shown in Fig. 2B, c. It can be seen that the chromosome number distribution changed from predominantly tetraploid to predominantly diploid. A number of intermediate values were also found. The isolation of S2 clones Samples from the seventh subculture of S2 in PRL4 with 1 mg/1. NAA were used to isolate clones of single-cell origin in an attempt to obtain clones of cells with different ploidies. Twenty such clones have been maintained and tested for (1) their ability to differentiate into a green tissue by development of chloroplasts on exposure to continuous light (500-1500 lux) and (2) their response to the medium which induced Differences between sycamore calluses Diploid (52) Tetraploid (104) • T _r 40 30 _ 20 - 10 - 43 1 '—i 1 l 1 i l i i i i l 1 1 1 1 B 30 o DO « 20 S 10 c wit 1 1 | I i 1—1 ! ! 11 C 11 £° 30 1 20 - I io 1 1 | | ^ 1 1 1 J , , | 1—<—1 1 1 D 30 - r-. 20 - uS 10 1 L/"> t^\ 1—1 to \o ^o r^. r*^ co co o^ o^ t L^l imm3 \f} t, ,> L^^ tmm3 Ify f*~^ LO ^ZJ * 1 1 1 II o "> Chromosome number range Fig. 2. The chromosome number distributions of sycamore tissues on different media (Table i ) ; A, S2 in its usual medium; B, after 5 subcultures in PRL4 with 1 mg/1. NAA; c, after 8 subcultures in PRL4 with 1 mg/1. NAA; and D, S4 in PRL4 with 1 mg/1. NAA. xylem and phloem formation in S4. Simpkins et al. (1970) have already reported that uncloned S2 callus can become green when exposed to light. All the clones showed a certain amount of greening after 8 weeks in the light but histological examination of the calluses grown for 6 weeks on PRL4 with 1 mg/1. NAA and 0-1 mg/1. kinetin did reveal differences in the type of growth which occurred. In most cases the cells of the callus showed little cell contact and many of the cells had broken up (Fig. 7), but 5 clones contained areas where the cells were in close contact with one another giving the callus an appearance more characteristic of S4 than S2 (Figs. 11, 12). These clones are being maintained on PRL4 with 1 mg/1. NAA. 44 K. Wright and D. H. Northcote The effect of polyethylene glycol on S2 callus The effect of the hormone ratios on S4 callus led us to consider a method of changing the texture of S2 callus. Thus we included polyethylene glycol (mol.wt.iooo) and sucrose in various combinations in a solidified medium based on PRL4 which also contained 1 mg/1. NAA and o-i mg/1. kinetin. The appearance of the callus after 6 weeks is shown in Fig. 5. Although 16 % polyethylene glycol appeared to inhibit the growth of the callus, other concentrations did not substantially affect the amount of growth which occurred. Histological examination of the tissue showed that it was the concentration of polyethylene glycol which affected the pattern of growth within the callus; over the concentration range used no visible effect of sucrose was noted. Calluses grown on media containing no polyethylene glycol were watery and did not stand up well to processing for cytological examination, and only occasional areas maintained their integrity to show well separated cells (Fig. 8). The inclusion of polyethylene glycol in the medium caused the calluses to be much harder, comparable with compact S4, and the cells were more closely packed together. This was most apparent with 16 % polyethylene glycol (Fig. 13), but many of the cells had been killed. At 4 and 8% polyethylene glycol the cells were also closely packed although they seemed less constrained to grow in rows than in the presence of 16 % polyethylene glycol (Fig. 14). Some areas of high mitotic activity occurred although no cellular differentiation was found in the calluses incubated on any of the media containing polyethylene glycol. DISCUSSION As in the experiments of Skoog & Miller (1957) the ratio of auxin to cytokinin was found to be the factor which controlled differentiation in S4. Roots were most often induced when the ratio was between 0-5 and 20 and usually at a stage when the calluses could not be classified as appearing white or compact. At low ratios of hormones the tissues were pigmented and very hard; xylem and phloem formation was observed although root formation was infrequent. At high ratios the calluses had a characteristic white colour and differentiation was found only once. It may be that the ratio of auxin to cytokinin was not the only controlling factor since ratios of 4 and 5 which usually induced root formation failed to do so when associated with 10 mg/1. NAA. The calluses on these media were white in appearance and in general showed much less differentiation. The increased pigmentation in the presence of kinetin may have been the result of the production of a secondary product from the tissue. Deposition of secondary material in the vacuole of S4 has been observed (Wright & Northcote, 1972) and this has recently been reported in S2 by Carceller, Davey, Fowler & Street (1971). Since no differentiation was found in S2 when it was cultured alongside S4 that was differentiating in the same flask S4 did not produce any diffusible extracellular watersoluble substance capable of inducing differentiation in S2 callus. Equally S2 did not produce any factors which were capable of inhibiting differentiation in S4 tissue and the reasons for the inability of S2 to differentiate must be sought elsewhere. Differences between sycamore calluses One obvious difference between the tissues S2 and S4 was the ploidy. The cells of S2 were mainly tetraploid while the new tissue S4 was predominantly diploid. Torrey (1958, 1961, 1967) found a similar variation with pea-root callus and as with the pea tissue the tetraploidy of S2 may have been induced by the cytokinins that were present in the coconut-milk. By transferring the S2 suspensions through a series of media with decreasing kinetin concentrations and finally to the one used to culture the diploid S4 (PRL4 with 1 mg/1. NAA) it has been possible to induce a population of predominantly diploid S2 cells. Although there are a number of differences between the media used for S2 and S4 (Table 1, p. 39) it is most probable that it was the difference in kinetin concentration that was responsible for this change in ploidy. It might have arisen by some kind of reduction division of the tetraploid cells or by mitosis and preferential selection of diploid cells that were present in the original culture in small numbers or that had reverted to diploid in the absence of kinetin. Clones have been isolated from such suspension cultures in order to try and isolate a pure diploid callus strain of S2 which will be tested for its ability to differentiate. So far 5 clones have been identified which have a growth pattern such that a greater number of cells are in contact with one another. If a tetraploid clone can also be isolated it will be possible to define more closely the mechanism of the reduction from tetraploid to diploid. In a reciprocal way tissues of S4 have been cultured on kinetin-containing media and have ceased to produce roots after 2 subcultures. It can be seen that the relationships of kinetin to polyploidy and differentiation are complex. Kinetin stimulates polyploid mitoses in pea root segments (Torrey & Fosket, 1970) and even in salamander tissue (Kevin, Witkus & Berger, 1966) and it is also known that many cells in the intact plant are polyploid (D'Amato, 1964). We have shown that kinetin acts on the predominantly diploid callus S4 to induce roots, whereas prolonged culturing in the presence of kinetin yields a tetraploid tissue that will not differentiate. A possible explanation is that the cells must undergo the transition from diploid to tetraploid (or higher degrees of polyploidy) under the correct conditions for differentiation to be induced as a result of the environmental factors controlling the transcription of the DNA. The requirement that cell division should immediately precede differentiation has been noted in plants (Fosket, 1968) and animals (Stockdale & Topper, 1966) and these authors suggest that it is the milieu in which cell division occurs that determines the pathway of development of the daughter cells. Thus polyploid cell formation may be a way of making the DNA susceptible to environmental control in the same way that this occurs at a normal cell division (Kauffman, 1967). Partanen (1965) has considered the function of polyploidy in the intact plant and argued that cells may become polyploid when the nutrient supply falls so low that the amount of energy available does not reach an envisaged predetermined level necessary for the discrete process of cytokinesis. DNA replication, being a more prolonged process, might continue slowly in the presence of this low amount of energy with the result that the cell would become polyploid. This theory neither indicates how the normal precise control mechanisms involved in correlating DNA replication and cytokinesis (Mather, 1965) could be overcome, nor does it explain the inducement of 45 46 K. Wright and D. H. Northfote polyploidy by kinetin. Although kinetin causes the cells to become smaller and more active it seems unlikely that this occurs to such an extent that the carbon supply becomes limiting. Cell division and differentiation are usually thought to be controlled by the ratio of the various hormones in the environment of the cell and possibly by the gradient of these substances across the cell. This may also be true of the control of polyploid cell formation. Auxins are synthesized by leaves (Jacobs, 1952) and cytokinins are produced by roots (Kende, 1965) and both of these hormones may be formed during the controlled autolytic processes of xylem and phloem formation (Wooding & Northcote, 1964; Northcote & Wooding, 1966; Sheldrake & Northcote, 1968). The ratio of these hormones at a particular point within a plant would depend on the physical dimensions of the plant as well as on other factors and thus such a ratio may differ from plant to plant. Conditions in some plants but not in others might favour polyploid cell formation during differentiation of certain tissues and in both cases there would appear to be the same potential for allowing re-programming of the DNA. The patterns of development which occur in the intact plant depend on cell-to-cell interactions. Cells in the plant make direct contact by means of plasmodesmata and there are common cell walls between adjacent cells. It is probable that the walls can act to carry material between cells and this function will vary according to the composition and developmental stage of the wall. The properties of the cell wall also determine the degree of mutual adhesion of the cells and this is especially important for the cell-to-cell interaction of callus tissues. Sycamore callus tissue cell walls contain relatively large amounts of pectic materials similar to cambial pectic substances (Stoddart, Barrett & Northcote, 1967; Stoddart & Northcote, 1967). A physiological low auxin concentration is usually thought to be required for differentiation to occur (Fosket & Roberts, 1964), and Rubery & Northcote (1970) have shown that under these conditions less arabinose is inserted in the neutral blocks and side chains of the pectin giving less branching and resulting in a tighter cell wall. Kinetin induces a harder texture in S4 and a certain amount of kinetin is required before differentiation can occur (Wright & Northcote, 1972). If the callus becomes too hard however root formation is uncommon; Torrey & Shigemura (1957) also found this using pea-root callus. The inclusion of polyethylene glycol in the medium causes the cells of S2 to come into greater contact and recently divided cells give the appearance of being physically constrained by their close neighbours. When Doley & Leyton (1970) applied polyethylene glycol to Fraxinus twigs a greater amount of xylem was formed in the wound callus which was also harder than in the absence of polyethylene glycol. Water can be considered a structural component of the plant cell wall and the amount of water available to pectin when it forms a gel can markedly affect the texture of the cell wall (Northcote, 1972). Thus it may be possible to increase the extent of cell interaction by either altering the chemical nature of the wall constituents, which occurs naturally during development, or by artificially regulating the amount of the water component which can be incorporated into the cell wall. Kochhar, Bhalla & Sabharwal (1971) have reported the induction of vegetative buds in tobacco callus by chelating agents and there is evidence to suggest that chelating Differences between sycamore calluses 47 agents cause a loosening of the wall by removing the calcium ions associated with the pectin (Xorthcraft, 1951). Thus the texture of the cell wall can be altered physically, chemically and hormonally and in all cases the pattern of differentiation in the tissue changes. It may be therefore that in order to induce differentiation of a callus tissue it is necessary to have not only genetically competent cells and the correct hormonal environment, but tissues of a texture compatible with allowing the appropriate cell-tocell interaction to take place. K. W. is grateful to the Science Research Council for a grant during the tenure of which this work was carried out. We would like to thank Mrs M. Wilson for typing the manuscript and Mr L. Jewitt and Mr R. 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Bot. 44, 334-344. WOODING, F. B. P. & NORTHCOTE, D. H. (1964). The development of the secondary wall of the xylem in Acer pseudoplatanus. J. Cell Biol. 23, 327-337. WRIGHT, K. & NORTHCOTE, D. H. (1972). Induced root differentiation in sycamore callus. J. Cell Sci. 11, 319-337. ZVVAR, J. A. & BRUCE, M. I. (1970). Cytokinins from apple extract and coconut milk. Aust. J. biol. Sci. 23, 289-297. {Received 11 April 1972) Differences between sycamore calluses Polyethylene glycol, % Figs. 3-5. For legend see p. 50. 49 K. Wright and D. H. Northcote Fig. 3. The difference in external appearance of white (top) and compact (bottom) S4 calluses. The white callus was friable and the compact one hard and pigmented. Callus diameter was about 2-5 cm. Fig. 4. Pieces of S4 callus surrounding a piece of S2 after 6 weeks growth together on solidified PRL4 with 1 mg/1. NAA and o-i mg/1. kinetin. Roots (arrows) are visible on some of the S4 calluses. Actual size. Fig. 5. The effect of sucrose and polyethylene glycol on S2 callus. Calluses grown in the presence of polyethylene glycol were similar in texture to the compact S4 shown in Fig. 3. Diameter of calluses grown in the presence of o, 4 and 8 % polyethylene glycol was about 2-5 cm. Fig. 6. A well developed area of xylem and phloem in a compact callus (S4). (Medium containing 0 8 mg/1. NAA, 2 mg/1. kinetin). The section was stained with aniline-blue and observed with ultraviolet fluorescence optics, x 440. Figs. 7—14 are sections of material embedded in wax and stained with safranin and picric aniline blue. Fig. 7. A cross-section through an area of an S2 clone in which the cells showed little intercellular contact. This was the normal growth pattern of S2 calluses, x 100. Fig. 8. Callus (S2) grown o n P R L 4 with img/1. NAA and 5-2 % sucrose, but no polyethylene glycol. The cells are well separated, x 100. Fig. 9. A cross-section through the surface area of a typical white callus (S4). The cells are in contact with one another but they are large and there is no organization, x 145. Fig. 10. A cross-section through a compact callus (S4). (Medium containing 0-5 mg/1. NAA, o-8 mg/1. kinetin.) There is more organization and the cells are very much smaller than in a white callus (Fig. 9). One nodular area containing xylem is encircled, x 140. Differences between sycamore calluses 4-? 52 K. Wright and D. H. Northcote Figs, i i , 12. Two different S2 clones isolated onPRL4with 1 mg/1. NAA in which the degree of cell contact is similar to that in S4 (Fig. 9) rather than to that in typical S2 (Fig. 7). Fig. 11, x 135; Fig. 12, x 200. Fig. 13. The surface region of an S2 callus grown in the presence of 1 6 % polyethylene glycol. Some of the cells appear to have been constrained to divide in radial columns (arrows), x 140. Fig. 14. S2 callus grown in the presence of 4 % polyethylene glycol. There is little intercellular space. Contrast with Fig. 8 where no polyethylene glycol was added, x 160. Differences between sycamore calluses 53