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J. Cell Set. II, 319-337 (1972) 319 Printed in Great Britain INDUCED ROOT DIFFERENTIATION IN SYCAMORE CALLUS K. WRIGHT AND D. H. NORTHCOTE Department of Biochemistry, University of Cambridge, Cambridge, CBz \QW, England SUMMARY A sycamore callus (S 4) has been isolated and grown on a medium containing salts, vitamins, a casein digest, 2 % sucrose and 1 mg/1. NAA. The callus, which would not grow in the absence of the added auxin, was much firmer in texture than a sycamore callus (S 2) isolated in this laboratory in 1958 which has not been induced to differentiate. When kinetin over the range 0-05-0-5 mg/1. was included in the growth medium of S 4 nodules of xylem and phloem were induced within the tissue and roots frequently grew from the surface of the callus. Some roots developed geotropic sensitivity although the majority grew radially outwards from the callus surface. The roots also varied with respect to the number of root hairs they carried. No roots were produced at sucrose concentrations less than 2%, although histological examination revealed extensive xylem and phloem differentiation relative to the amount of growth which had taken place. When sugars other than sucrose were supplied in the medium at a concentration of 3 % ( w / v ) roots were also induced in those calluses where the carbon source had supported good growth. Sucrose, glucose and fructose were identified in the ethanol-soluble extracts of all these calluses. Radioactivity was incorporated into sucrose when S 4 was incubated on a medium con- taining D-[U- 14 C]glucose for 24 h. Any sugar which supported growth and differentiation was therefore one which was capable of entering the common metabolic pathway used by the plant for glucose and sucrose. The cells could undergo differentiation so long as the sugar they were supplied with supported active growth and division. The possibility of a physiological role for sucrose is discussed. INTRODUCTION Plant callus tissues have been a valuable tool in the investigation of the factors that control differentiation. They are particularly suitable for such work, since the tissue can be equilibrated under one set of conditions and then transferred to another so that the change has clearly been responsible for the events which occur after the transfer. When tissue excised from an intact plant is used the endogenous growth factors and the mechanism for their transport are often unknown and therefore cannot be controlled experimentally. Numerous callus tissues have been used in investigations of this kind (Roberts, 1969; Halperin, 1969). Usually xylem elements are formed, although many tissue cultures will also differentiate to form phloem, some may form roots and there is an increasing number of tissue cultures which can give rise to intact plants. The differ- entiation is almost always a result of the manipulation of the levels of auxin and cytokinin in the growth medium. However it is not always possible to induce differ- entiation in a callus tissue, either because the correct balance of hormonal and nutri- tional factors has not been found or because the cells fail to respond to such conditions.

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Page 1: INDUCED ROOT DIFFERENTIATION IN SYCAMORE CALLUS · INDUCED ROOT DIFFERENTIATION IN SYCAMORE CALLUS K. WRIGHT AN DD. H. NORTHCOTE Department of Biochemistry, ... of the callus grown

J. Cell Set. I I , 319-337 (1972) 319

Printed in Great Britain

INDUCED ROOT DIFFERENTIATION IN

SYCAMORE CALLUS

K. WRIGHT AND D. H. NORTHCOTE

Department of Biochemistry, University of Cambridge, Cambridge, CBz \QW, England

SUMMARY

A sycamore callus (S 4) has been isolated and grown on a medium containing salts, vitamins,a casein digest, 2 % sucrose and 1 mg/1. NAA. The callus, which would not grow in the absenceof the added auxin, was much firmer in texture than a sycamore callus (S 2) isolated in thislaboratory in 1958 which has not been induced to differentiate. When kinetin over the range0-05-0-5 mg/1. was included in the growth medium of S 4 nodules of xylem and phloem wereinduced within the tissue and roots frequently grew from the surface of the callus. Some rootsdeveloped geotropic sensitivity although the majority grew radially outwards from the callussurface. The roots also varied with respect to the number of root hairs they carried. No rootswere produced at sucrose concentrations less than 2 % , although histological examinationrevealed extensive xylem and phloem differentiation relative to the amount of growth which hadtaken place. When sugars other than sucrose were supplied in the medium at a concentration of3 % (w/v) roots were also induced in those calluses where the carbon source had supported goodgrowth. Sucrose, glucose and fructose were identified in the ethanol-soluble extracts of all thesecalluses. Radioactivity was incorporated into sucrose when S 4 was incubated on a medium con-taining D-[U-14C]glucose for 24 h.

Any sugar which supported growth and differentiation was therefore one which was capableof entering the common metabolic pathway used by the plant for glucose and sucrose. The cellscould undergo differentiation so long as the sugar they were supplied with supported activegrowth and division. The possibility of a physiological role for sucrose is discussed.

INTRODUCTION

Plant callus tissues have been a valuable tool in the investigation of the factors thatcontrol differentiation. They are particularly suitable for such work, since the tissuecan be equilibrated under one set of conditions and then transferred to another sothat the change has clearly been responsible for the events which occur after thetransfer. When tissue excised from an intact plant is used the endogenous growthfactors and the mechanism for their transport are often unknown and therefore cannotbe controlled experimentally.

Numerous callus tissues have been used in investigations of this kind (Roberts,1969; Halperin, 1969). Usually xylem elements are formed, although many tissuecultures will also differentiate to form phloem, some may form roots and there is anincreasing number of tissue cultures which can give rise to intact plants. The differ-entiation is almost always a result of the manipulation of the levels of auxin andcytokinin in the growth medium. However it is not always possible to induce differ-entiation in a callus tissue, either because the correct balance of hormonal and nutri-tional factors has not been found or because the cells fail to respond to such conditions.

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320 K. Wright and D. H. Northcote

Acer pseudoplatanus callus, maintained in this laboratory since it was isolated here14 years ago (Lamport & Northcote, i960) has not been stimulated to differentiate(Torrey, 1968) although Simpkins, Collin & Street (1970) have obtained greening ofthe tissue under certain conditions. It has been distributed to a number of laboratoriesand has been used for a variety of studies. As far as we know, there are no other isolatesof callus from A. pseudoplatanus in use. Simola & Sopanen (1970) and Scala &Semersky (1971), have used this culture to study the activity of certain enzymes.Roberts & Northcote (1970 a, b) have used the culture in ultrastructural studies ofcell division and of nuclear pore structure. The composition and formation of thecell wall have also been studied (Lamport & Northcote, i960; Stoddart, Barrett &Northcote, 1967; Stoddart & Northcote, 1967; Rubery & Northcote, 1970; Heath &Northcote, 1971). In addition, Lescure (1969) has selected auxin-independent mutantsfrom cultures of the tissue, and Street and his colleagues have made a detailed studyof the callus grown as a suspension culture concentrating mainly on its growth andmetabolism (Street, Collin, Short & Simpkins, 1968; Davey & Street, 1971).

Although the tissue is still an ideal material for studies on growth and division, itsinability to differentiate limits its experimental value. As part of a wider study, freshtissue cultures of A. pseudoplatanus have been induced and compared with the originalcallus. We report here the properties of one particular fresh isolate and its response todifferent concentrations of kinetin and auxin and to a variety of carbon sources.

MATERIALS AND METHODS

Tissue culture

All chemicals used throughout this study were of A.R. grade wherever possible. The mediumused was the PRL 4 medium of Gamborg (1966); it contains no coconut milk and is completelydenned except that it contains a casein digest. In the text 'PRL 4' indicates a medium that con-tains inorganic salts, the casein digest and vitamins in the concentrations used by Gamborg.Callus was routinely maintained on PRL 4 with 1 mg/1. NAA (i-naphthylacetic acid) and 2%sucrose. Media were made up using glass-distilled water, solidified using 1 % (w/v) Agar Noble(Difco Laboratories, Detroit, Michigan, U.S.A.), and then sterilized together with instrumentsand apparatus by autoclaving at 103-4 kN m~* (15 lb/in.1), 120 °C for 30 min. Sterile operationswere carried out in a cabinet fitted with rubber gloves and an ultraviolet lamp that was switchedon for 20 min before using the cabinet. Tissue cultures were incubated at 26 ± 2 °C in the darkin boiling tubes (25 mm diameter) or in sterile plastic jars (Richardsons of Leicester Ltd,Leicester, U.K.). If a large number of different media was required these were made up andautoclaved in the tubes whereas a number of identical replicates was obtained by pouringautoclaved molten agar into individual sterile jars.

Initiation of calluses

During a warm period in January (1970) germinating seeds were collected from beneath anisolated sycamore tree and planted in moist vermiculite in a greenhouse. In May (1970) thehypocotyls were removed, sterilized in Milton solution (Milton Division, Richardson-MerrellLtd, London, W. 1, U.K.) for 15 min, washed twice in sterile glass-distilled water, and then cutinto approximately 10-mm lengths with a sterile scalpel. The explants were laid horizontally onsolidified PRL 4 with 1 mg/1. NAA and 2 % sucrose.

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Root differentiation in sycamore callus 321

Microscopy

Tissue was processed for light microscopy by embedding in either wax or glycol methacrylate.For wax embedding the tissue was fixed in methanol/chloroform/propionic acid (6:3:2) fixativeovernight, dehydrated in 3 or 4 changes of 2-ethoxy-ethanol (ethyl cellosolve), transferred to2-methyl-propan-2-ol at 30 CC and then finally infiltrated with and embedded in Paraplast wax(m.p. 56-57 °C) (Sherwood Medical Industries Inc., St Louis, U.S.A.). Sections were cut at5 fim on a Jung rotary microtome, floated on to gelatine-coated slides, cleared, and then stainedwith safranin followed by picric aniline-blue (Jensen, 1962). Phloem was detected by ultra-violet fluorescence microscopy of aniline-blue stained sections (Currier & Strugger, 1956). Theprocedure for embedding in glycol methacrylate and cutting and staining sections has beendescribed by Jones & Northcote (1972). Sections were mounted on slides in DPX mountant(G. T. Gurr and Co. Ltd., London, England) and examined using a Zeiss Ultraphot, Model II.Xylem was detected most easily with polarized light.

Incubation with sugars other than sucrose

The medium used was PRL 4 with 1 mg/1. NAA and with the alternative sugars used at 3 or5 %. The purity of the sugars used was checked by descending paper chromatography usingethylacetate/pyridine/water (8:2:1) as described by Harris & Northcote (1970); this systemwas also used for the separation of sugars extracted from the calluses. Spots were developedusing the silver nitrate-sodium hydroxide method of Trevelyan, Procter & Harrison (1950) orsprayed with />-amino-phenol reagent (Vamos, 1953). A sample of D-[U-"C]glucose (TheRadiochemical Centre, Amersham, Bucks., U.K.) was also purified by separation on thischromatographic system and subsequent elution.

When it was necessary to avoid autoclaving the sugars they were omitted from the medium tobe autoclaved and the sugar solution was filter-sterilized using a Millipore apparatus and filterof pore size 045 /im (Millipore (U.K.) Ltd., Abbey Road, London, N.W. 10) and then addedjust before the agar set.

Inocula were small in order to minimize the transfer of material from the previous medium.An approximate 0-5-cm cube of the inoculum was transferred to 20 ml of the medium. Afterthe callus had been incubated on a particular carbon source for the required time it was removedfrom the surface of the agar (no agar was carried with it) and homogenized in ethanol using apestle and mortar (Ball, 1955). The solids were removed by filtration and the ethanol wasreduced by rotary evaporation before application to the chromatogram. Sucrose was hydrolysedby incubation in o-i N HC1 for 2 h at 100 °C and subsequent neutralization. Chromatogramswere processed for scintillation counting as described by Harris & Northcote (1970). Eachsample was counted for 10 min 4 times.

RESULTS

Properties of the callus

Six days after the hypocotyl segments were explanted on to PRL 4 with 1 mg/1.NAA and 2 % sucrose, callus began to develop all along the explant and also formed awedge at the ends (Fig. 2). Sections of fixed, embedded tissue showed that the callushad a mixed origin. Cortical cells expanded and divided to break the epidermis andsmall meristematic cells with prominent nuclei proliferated to form the wedge at theends of the explant (Fig. 5). Roots were sometimes formed on the original explant(Fig. 3) but these were never seen after the first subculture.

Of the callus tissues formed from the explants set up, 8 are being maintained at thepresent time. Of these one in particular designated S4 has been selected and is thecallus used in all the experiments reported here. The original isolate (1958) is desig-nated S2. Callus S4 that had been grown on PRL4 with 1 mg/1. NAA and 2%

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322 K. Wright and D. H. Northcote

sucrose was much less friable than S2 cultured under the usual laboratory conditions(as described for Phaseolus vulgaris by Jeffs & Northcote, 1966). It was subculturedevery 3-4 weeks and had been taken through 16 transfers by August 1971. It grew onsolid media in a hemispherical form and proliferation occurred most rapidly justbelow the upper surface of the callus.

On the inside of the tonoplast of some cells hemispherical deposits were visiblewhich stained bright red-purple with safranin/picric aniline-blue (Fig. 6). Theywere only found in fixed, embedded tissue and have not been seen in live cells. Inlarger quantities these deposits filled the whole vacuole (Figs. 4, 14) and the tissuewas then difficult to embed. The substance may be actively deposited in certain cells,or it may be a product of cell death; in any case it is presumed to be some kind ofsecondary product. Goldstein, Swain & Tjhio (1962) have shown that leucoantho-cyanins can be found in cultures of S2, and using the method of Swain & Hillis(1959) it has been confirmed that this is also true of S4.

The cells of S4 were typical tissue culture cells exhibiting the usual range of shapesand sizes and showing active cytoplasmic streaming as described for S2 by Roberts &Northcote (1970a). The nucleus was either against the wall or suspended by cyto-plasmic strands in the centre of the vacuole. Usually the nucleus had only one nucleo-lus, but nuclei with 2 or more nucleoli and also cells with 2 nuclei were occasionallyseen.

Dependence of the callus on NAA

The concentration of NAA in the medium was varied in order to see to whatextent the callus was dependent on this auxin. Fig. 7 shows the growth obtained after3 weeks 4 days of the third transfer on medium containing different amounts of NAA;one of the 3 pieces of callus employed in the fresh weight determination is used tomark on Fig. 7 the extent of growth. Essentially the same pattern of growth wasobtained after the same period of the second transfer, so that the extent of growthof the callus was shown to be dependent on the amount of supplied auxin. Histo-logical examination showed that a small amount of xylem differentiation was presentin the callus supplied with o-oi mg/1. NAA. S4 callus did not grow on media con-taining up to 20 mg/1. IAA (indol-3yl acetic acid), presumably due to the action ofoxidases produced by the tissue.

Effect of kinetin

Under the routine conditions for growth of S 4 all the cells were of the parenchymat-ous nature described above, and, with one exception (see later), no differentiationwas found in prepared sections of fixed and embedded tissue. In contrast, however,when kinetin (6-furfuryl-amino-purine) was added to the culture medium and theother conditions were maintained as before (NAA at 1 mg/1., sucrose at 2 %) extensivedifferentiation occurred within the callus and roots were frequently produced (Fig. 8).This was observed with kinetin over the concentration range 0-02-2-0 mg/1. althoughit occurred more frequently over the range 0-05-0-5 mg/1. This was repeated a numberof times and while, for a given concentration of kinetin a root was not produced every

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Root differentiation in sycamore callus 323

time, sectioning of replicates which had not produced roots always revealed extensivedifferentiation consisting both of well separated areas of xylem and phloem and com-pact nodules of the type found by Jeffs & Northcote (1966) in Phaseolus tissue (Figs.14-18).

The roots appeared after 3-4 weeks and they carried different numbers of root hairs;they also varied in their capacity to display geotropism. In the early stages, the rootsseemed to be linear extensions of the spherical nodule which gave rise to themand the callus tended to grow radially outwards from an area just below the surface.The roots always began their growth at right angles to the surface at the point atwhich they occurred and many of them continued to do so until their own weightcaused them to turn downwards. Less commonly, roots began to grow downwardsimmediately and there were also intermediate stages. Fig. 11 shows a root which hadbent through a right angle to grow downwards and penetrated the agar medium.Usually roots showing geotropism were formed on the same medium as those notdoing so, so that this variation did not depend on any constituent of the medium.

A root cap on a root 4 mm long which was not responding to gravity is shown inFig. 13 and Fig. 12 shows a cross-section of the same root. The triarc pattern of thestele was consistent with the triarc patterns often found in young roots of comparablelength obtained from sycamore seedlings.

Hairs may be seen arising from the epidermal cells (Fig. 12) and they may com-pletely cover the root (Fig. 10), or they may occur locally along the root (Fig. 11) or beabsent altogether (Fig. 9). It may be that the development of root hairs is influencedby the humidity in the immediate vicinity of the root and, like the geotropic response,the development of root hairs cannot be attributed to the chemical composition of themedium.

Effect of different kinetin and sucrose levels

The tissues were incubated in PRL4 with 1 mg/1. NAA and the concentration ofkinetin was varied over the range 0-05-2-0 mg/1. For each concentration of kinetin,sucrose was varied from 0-25 to 5 %. The results are shown in Fig. 21 and the arrowsshow the positions of roots. Irrespective of the kinetin concentration, no roots wereformed at sucrose concentrations of 1 % or less but at 2 % or above they were pro-duced frequently.

Calluses which had not produced roots were fixed, embedded and sectioned inorder to investigate the extent to which differentiation had occurred. Sections ofcallus grown on 1 % sucrose were, in many areas, indistinguishable from sectionsgrown on 2 % sucrose and xylem and phloem tissue were identified both in thesespecimens (Figs. 19, 20) and to a lesser extent in calluses grown on 0-5% sucrose.No differentiation was found in tissue grown on 0-25 % sucrose where most of thecells had died.

Effect of different carbon sources

The growth and differentiation of the tissue. In order to investigate any possiblemorphogenetic response of the callus to specific sugars, a number of mono-, di-, and

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324 K. Wright and D. H. Northcote

Table i. The effect of various sugars on the growth and differentiation of S4 after8 weeks

(Sugars found in ethanolic extracts of the callus are also shown, and the same ones were detectedwhether or not kinetin had been included in the medium.)

Sugar Growth Diffn Soluble sugars identified in callus extractfed to , * N , A >callus — kinetin + kinetin + kinetin Six Glu Fru Gal Man Cel Lac Mai Tre Raf

D-G/ucose • • • • • • Roots • • • O O O O O O OD-Frwctose • • • • • Roots • • • O O O O O O O

• • • O O O O O O OD-Ga/actose • • None found — — — — — — — — — —n-Mannose • • • • • Roots • • • O » O O O O OSucrose • • • • • • • Roots • • • O O O O O O OCWlobiose • • • • • • Roots • • • 0 0 * 0 0 0 0Lactose D H None found — — — — — — — — — —Maltose • • • xyl+phl • • • O O O O « O OCLO. Trehalose • • • • • • • Roots • • • O O O O O 9 Oito/finose • • • • • • • Roots • • • • O O O O O •

The number of squares ( • ) indicates extent of growth of the callus; • , no growth; • , detec-tion of sugar; O, the sugar not detected; —, callus not analysed; xyl + phl, xylem + phloem.

trisaccharides were tested for their ability to sustain growth and differentiation. Themedia used consisted of PRL4 with 1 mg/1. NAA and o-i mg/1. kinetin, with thesugar at a concentration of 5 % if the medium was sterilized by autoclaving or 3 %if Millipore filtration was used. Kinetin was omitted from the controls. Five replicatesof each incubation were set up and inspected periodically during the incubationperiod of 8 weeks. Both autoclaved and sterile-filtered sugars were used and the resultsof the latter experiment are shown in Table 1. When the carbon source was not capableof supporting callus growth the inclusion of kinetin in the medium caused some slightgrowth over the 8-week period. When the callus grew well the addition of kinetin tothe medium invariably caused a decrease in the overall size of the callus (Fig. 22a-d)and the individual cells were smaller.

From Table 1 it is clear that the media which supported good growth of the callusin the presence of kinetin were also those which allowed root formation. Wherelimited growth had occurred either with low concentrations of sucrose or when maltosewas supplied, xylem and phloem were identified but no roots were formed. No dif-ferentiation was observed in those calluses which failed to grow.

In all experiments except one, kinetin was necessary for differentiation. A nodulecontaining xylem and phloem within an isolated area of the callus was found in anincubation containing raffinose but no kinetin. Since the callus presumably synthe-sizes some endogenous cytokinin, local conditions of cytokinin synthesis may havebeen such as to allow differentiation to occur in this experiment. Raffinose was a very

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Root differentiation in sycamore callus

Sucrose Glucose Fructose

325

6 8 10 12 14 16 18 20 22 24 26 28 30 32 34

Fraction number

Fig. 1. The pattern of labelling found in ethanolic extracts of callus fed D-[U-14C]glu-cose after chromatographic separation on paper using ethyl acetate: pyridine: water(8:2:1 by vol.). A, distribution before hydrolysis. Fractions 4-9 from A were combinedand hydrolysed and the distribution of radioactivity is shown in B. The positions ofsucrose, glucose and fructose markers are indicated.

good carbon source for growth so it may be that the conditions were particularlyfavourable for differentiation.

The only difference found between incubations using sterile filtered sugars andthose using autoclaved sugars was found for fructose; when the autoclaved sugarwas used, less growth occurred than when the sugar was sterile filtered. Stehsel &Caplin (1969) have observed that autoclaved fructose and glucose were inhibitoryto the growth of carrot tissue cultures.

TJie neutral sugar pool. The free sugars present in the callus were examined afterdifferentiation had occurred. The results are summarized in Table 1 and show thatsucrose, glucose and fructose were formed from all the carbon sources (autoclaved orsterile filtered) on which the callus grew since these sugars were always found in theextracts of the tissue. Only the sugar that had been supplied to the inoculum waspresent in extracts of callus incubated on media that had not supported growth andthus the sugars detected were not those transferred with the original inoculum.

Solid pieces of S4 callus were placed in a liquid incubation medium containing onlyD-[U-14C]glucose (0-05 /tCi) as a carbon source for 24 h, an ethanol extract was madeand a sample of the extract was applied to a chromatogram. The pattern of labellingof the sugars on the chromatogram showed a peak in the position of sucrose (Fig. 1 A)which, after hydrolysis, neutralization and rechromatographing in the same solventsystem, separated to occupy the positions of glucose and fructose (Fig. 1 B). Thussucrose had been synthesized from the supplied glucose.

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326 K. Wright and D. H. Northcote

DISCUSSION

Most tissue cultures, apart from certain mutants (Fox, 1963; Lescure, 1969),require added auxin for growth, and S4 is no exception. A cytokinin is not required,however, and if it is assumed that a cytokinin must be present for cell division to occur,then it is likely that the callus can make its own either by a synthetic process or as theproduct of autolysis in the same way that auxins may be produced (Sheldrake &Northcote, 1968 a-c).

Addition of kinetin to the medium shows that this is the factor limiting morpho-genesis under the conditions which we routinely used to grow the tissue. Fosket &Torrey (1969) found this to be true also of soybean callus; they reported that a lowerlimit of kinetin was required for tracheary element formation. We found a smallamount of xylem formation in the absence of kinetin, when only o-oi mg/1. NAA wassupplied. Skoog & Miller (1957) showed that the ratio of auxin to cytokinin was thefactor controlling differentiation in tobacco callus tissue, and it may be that theendogenous cytokinin content of S4 under the conditions used by us was such as toafford the correct ratio with the supplied auxin at o-oi mg/1.

It has not been possible to control the geotropic response of the roots produced inresponse to o-i mg/1. kinetin. Although most of the roots grew radially outwards fromthe surface of the callus, some have been observed to display positive geotropism.The control of the geotropic response in maize has been shown to depend on thepresence of a root cap (Juniper, Groves, Landau-Schachar & Audus, 1966). Itappears that the heavy starch grains respond to the gravitational force and affect thetransport of water-soluble substances that are produced by the root cap and whichreach the meristem by passage through the plasmodesmata (Iversen, 1969; Juniper &French, 1970; Iversen & Larsen, 1971; Pilet, 1971).

This root-producing system has been used to investigate the effect of sugars otherthan sucrose on the differentiation process. The possible physiological effects ofsucrose have been investigated by a number of workers, and the work is reviewed byRoberts (1969") and Torrey, Fosket & Hepler (1971). Using the classification of thelatter authors the effects of sucrose can be attributed to the following, (a) Its role as acarbon source. Growth and differentiation are intimately connected, and a factorwhich limits growth may be expected to limit differentiation, (b) Its osmotic effects(Doley & Leyton, 1970), although this may not be a primary action (Torrey et al.1971). (c) A possible 'hormone-like' function. Jeffs & Northcote (1967) found thatPhaseolus vulgaris callus tissue had a very specific carbon source requirement beforenodules containing both xylem and phloem were induced.

Torrey et al. (1971) have observed that it is not possible to find evidence in theliterature to confirm or deny the possibility that sucrose acts via its osmotic properties.Doley & Leyton (1970) found that as the water potential was decreased by increasingconcentrations of sucrose the callus growth on explants of Fraxinus changed from afriable, loosely packed group of cells with few tracheary elements to a hard mass withabundant tracheary elements. It is possible to increase the amount of differentiationin explants of Populus deltoides by applying a physical constraint to the proliferating

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Root differentiation in sycamore callus 327

cells (Brown, 1964), and if these observations are due to pressure effects as suggestedby the authors it may be that enhanced cell-cell interaction due to the imposed con-straints is of importance. In one experiment we have varied the sucrose concentrationover the range 0-25-5 % a t different levels of kinetin. The calluses grown on 5 %sucrose were larger and less friable than those grown on 0-25 % sucrose where therewere more dead cells whose cytoplasmic contents when released made the calluswatery. The osmotic properties of sucrose may also have some effect on the aboveresults (Doley & Leyton, 1970).

Callus S4 was far less specific in its carbon source requirement for phloem forma-tion than was Phaseolus vulgaris tissue (Jeffs & Northcote, 1967). Differentiation ofxylem and phloem occurred when any sugar was used on which good growth wasobtained. These calluses were examined to see which sugars were present in the tissuewhen sugars other than sucrose were supplied. Sucrose was synthesized not onlyfrom glucose but from all the substrate sugars including those on which no xylem orphloem differentiation was found and on which only moderate growth occurred. Thesimplest interpretation is that sucrose is important only as a carbon source, and thatif a different sugar is a sufficiently good carbon source, then it will be possible toobtain differentiation. The results obtained by various workers can be explained byassuming that the diverse tissues have dissimilar metabolic specificities with respectto the carbon source supplied.

Since sucrose is the normal substrate supplied to a cell in an intact plant the meta-bolism is adapted to utilizing this carbon source so that all the enzymes required for itsuptake and metabolism are present. In the short term, therefore, the availability ofthe sugar for use by the plant probably depends on the degree of its similarity tosucrose, and on the ease with which it can be acted upon by the enzymes alreadypresent. In the long term, however, the response to a particular sugar is more likely tobe affected by the ability of the tissue to produce enzymes to deal with it. If it cannottake up and subsequently metabolize the sugar, the cell will not grow or differentiate.When a sugar is only inefficiently used as a carbon source although the callus mayachieve a moderate growth, differentiation does not occur; this may be because thereis an insufficient supply of an intermediate substance that is required for normalgrowth associated with cell division and differentiation. Since differentiation mustproceed to a fairly late stage to be recognizable with the light microscope it is possiblethat initiation of xylem or phloem formation does occur, but that the subsequentmetabolic demands of the process on, for instance, the production of cell-wall com-ponents or their precursors are too great and the process aborts.

Sucrose, glucose and fructose were produced from any sugar which supportedgrowth and this has also been shown by Ball (1955). This indicates the importance ofthese sugars for growth and differentiation. Cell regulatory mechanisms may be suchas to necessitate the formation of sucrose as an intermediate in the metabolism of aparticular carbon source, and the ability of the callus to synthesize enzymes capableof this formation might determine the suitability of such a carbon source for growth.Such an interpretation is consistent with the results of Jeffs & Northcote (1967),Wetmore & Rier (1963) and Rier & Beslow (1967). Only when a sugar was supplied

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328 K. Wright and D. H. Northcote

that could be metabolized by the callus would there develop the area of active growthand cell division that seems necessary before cell differentiation can occur (Fosket,1968). However, it is more difficult to explain why phloem formation is favoured whenrelatively high sucrose concentrations are supplied and that xylem formation occursat lower concentrations (Wetmore & Rier, 1963; Jeffs & Northcote, 1966). It may bethat the energetic and synthetic requirements for the differentiation of phloem ele-ments are greater than those for xylem formation.

Sucrose may be synthesized by 2 enzymic reactions, (a) Sucrose phosphate synthe-tase (UDP-glucose:D-fructose-6-phosphate-2-glucosyltransferase, E.C. 2.4.1.14)catalysing the reaction

UDP-glucose + D-fructose-6-phosphate >sucrose-6-phosphate + UDP.Sucrose phosphate is then hydrolysed to give sucrose, (b) Sucrose synthetase (UDP-glucose :D-fructose-2-glucosyltransferase, E.C. 2.4.1.13) catalysing the reactionUDP-glucose+D-fructose-< >sucrose-|-UDP.

It is generally considered (Hassid, 1967; Hawker, 1971) that the first enzyme is theone which operates in vivo and which is responsible for the sucrose that is formed as aconsequence of photosynthesis. The latter enzyme is thought to perform a degradativefunction in vivo. Sucrose may also be degraded by 2 different invertases (/?-D-fructo-furanoside fructohydrolase, E.C. 3.2.1.26) which occur with acid and alkaline pHoptima (Ricardo & ap Rees, 1970). The amounts of these 4 enzymes have been foundto vary in different tissues and in different parts of the same tissue and the amount ofsucrose present can be correlated with the levels of these enzymes (Lyne & ap Rees,1971; Hawker, 1971). Hence there is a fine control over the concentration of sucrose,both intracellularly and extracellularly. In our experiments we have shown that in thecallus tissue sucrose is degraded to glucose and fructose when sucrose is fed and thatit is also synthesized since radioactive sucrose is found in the extracts of callus fedradioactive glucose. Hence local conditions in the callus may control variations in thesucrose level, with the possibility that this might exert some physiological effect.It is therefore very difficult to distinguish an essential nutritional requirement from ahormone. Edelman & Hanson (1971a, b) have shown that sucrose can suppresschlorophyll synthesis in a carrot callus tissue containing no invertase, whereas thiseffect was not observed using a different callus strain that had a high invertase activity.Mixtures of glucose and fructose were not capable of substituting for sucrose in thissystem and therefore it appears that sucrose acts in a specific way on this particulardevelopmental process. Nevertheless, since sucrose might be regarded as the end-product of chloroplast metabolism the control of chlorophyll synthesis may be by afeedback mechanism. We have observed that chlorophyll synthesis is not inhibited bysucrose in S4 and S2 since chloroplast formation has been observed in light-growncultures supplied with 2 % sucrose.

K. W. thanks the Science Research Council for a studentship during the tenure of which thiswork was carried out. We would also like to thank Mr L. Jewitt and Mr R. Pilcher for assistancewith the photography and Mrs M. Wilson for typing the manuscript.

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BROWN, C. L. (1964). The influence of external pressure on the differentiation of cells andtissues cultured in vitro. In The Formation of Wood in Forest Trees (ed. M. H. Zimmermann),pp. 389-404. New York and London: Academic Press.

CURRIER, H. B. & STRUGGER, S. (1956). Aniline blue and fluorescence microscopy of callose inbulb scales of AlUum cepa L. Protoplasma 45, 552-559.

DAVEY, M. R. & STREET, H. E. (1971). Studies on the growth in culture of plant cells. IX.Additional features of the fine structure of Acer pseudoplatanus L. cells cultured in suspension.J. exp. Bot. 22, 90-95.

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EDELMAN, J. & HANSON, A. D. (1971a). Sucrose suppression of chlorophyll synthesis in carrotcallus cultures. Planta 98, 150-156.

EDELMAN, J. & HANSON, A. D. (19716). Sucrose suppression of chlorophyll synthesis in carrottissue cultures: the role of invertase. Planta 101, 122-132.

FOSKET, D. E. (1968). Cell division and the differentiation of wound-vessel members in culturedstem segments of Coitus. Proc. natn. Acad. Sci. U.S.A. 59, 1089-1096.

FOSKET, D. E. & TOKREY, J. G. (1969). Hormonal control of cell proliferation and xylemdifferentiation in cultured tissues of Glycine max var. Biloxi. PL Physiol., Lancaster 44,871-880.

Fox, J. E. (1963). Growth factor requirements and chromosome number in tobacco tissuecultures. Physiologia PI. 16, 793-803.

GAMBORG, O. L. (1966). Aromatic metabolism in plants. II. Enzymes of the shikimate pathwayin suspension cultures of plant cells. Can. J. Biochem. 44, 791-799.

GOLDSTEIN, J. L., SWAIN, T. & TJHIO, K. H. (1962). Factors affecting the production of leuco-anthocyanins in sycamore cambial cell cultures. Archs Biochem. Biophys. 98, 176—177.

HALPERIN, W. (1969). Morphogenesis in cell cultures. A. Rev. PL Physiol. 20, 395-418.HARRIS, P. J. & NORTHCOTE, D. H. (1970). Patterns of polysaccharide biosynthesis in differ-

entiating cells of maize root-tips. Biochem. J. 120, 479-491.HASSID, W. Z. (1967). Transformation of sugars in plants. A. Rev. PL Physiol. 18, 253-280.HAWKER, J. S. (1971). Enzymes concerned with sucrose synthesis and transformations in seeds

of maize, broad bean and castor bean. Phytochemistry 10, 2313-2322.HEATH, M. F. & NORTHCOTE, D. H. (1971). Glycoprotein of the wall of sycamore tissue-

culture cells. Biochem. J. 125, 953-961.IVERSEN, T.-H. (1969). Elimination of geotropic responsiveness in roots of cress {Lepidium

sativum) by removal of statolith starch. Physiologia PL 22, 1251-1262.IVERSEN, T.-H. & LARSEN, P. (1971). The starch statolith hypothesis and the optimum angle of

geotropic stimulation. Physiologia PL 25, 23-27.JEFFS, R. A. & NORTHCOTE, D. H. (1966). Experimental induction of vascular tissue in an

undifferentiated plant callus. Biochem. J. 101, 146-152.JEFFS, R. A. & NORTHCOTE, D. H. (1967). The influence of indol-3yl acetic acid and sugar on

the pattern of induced differentiation in plant tissue culture. J. Cell Sci. 2, 77-88.JENSEN, W. A. (1962). Botanical Histochemistry. San Francisco and London: W. H. Freeman.JONES, M. G. K. & NORTHCOTE, D. H. (1972). Nematode induced syncytium - a multinucleate

transfer cell. J. Cell Sci. 10, 789-809.JUNIPER, B. E. & FRENCH, A. (1970). The fine structure of the cells that perceive gravity in the

root tip of maize. Planta 95, 314-329.JUNIPER, B. E., GROVES, S., LANDAU-SCHACHAR, B. & AUDUS, L. J. (1966). Root cap and the

perception of gravity. Nature, Lond. 209, 93-94.LAMPORT, D. T. A. & NORTHCOTE, D. H. (i960). Hydroxyproline in primary cell walls of

higher plants. Nature, Lond. 188, 665-666.LESCURE, A.-M. (1969). Mutagenese et selection de cellules d'Acer pseudoplatanus L. cultivees in

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LYNE, R. L. & AP REES, T. (1971). Invertase and sugar content during differentiation of roots ofPisum sativum. Phytochemistry 10, 2593—2599.

PILET, P. E. (1971). Root cap and georeaction. Nature, New Biol. 233, 115-116.RICARDO, C. P. P. & AP REES, T. (1970). Invertase activity during the development of carrot

roots. Phytochemistry 9, 239-247.RIER, J. P. & BESLOW, D. T. (1967). Sucrose concentration and the differentiation of xylem in

callus. Bot. Gas. 128, 73-77.ROBERTS, K. & NORTHCOTE, D. H. (1970a). The structure of sycamore callus cells during

division in a partially synchronized suspension culture. J. Cell Sci. 6, 299—321.ROBERTS, K. & NORTHCOTE, D. H. (19706). Structure of the nuclear pore in higher plants.

Nature, Lond. 228, 385-386.ROBERTS, L. W. (1969). The initiation of xylem differentiation. Bot. Rev. 35, 201-250.RUBERY, P. H. & NORTHCOTE, D. H. (1970). The effect of auxin (2,4-dichlorophenoxyacetic

acid) on the synthesis of cell wall polysaccharides in cultured sycamore cells. Biochim.biophys. Acta 222, 95-108.

SCALA, J. & SEMERSKY, F. E. (1971). An induced fructose-1,6-diphosphatase from cultured cellsof Acer pseudoplatanus (English sycamore). Phytochemistry 10, 567-570.

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STODDART, R. W., BARRETT, A. J. & NORTHCOTE, D. H. (1967). Pectic polysaccharides ofgrowing plant tissues. Biochem. J. 102, 194-204.

STODDART, R. W. & NORTHCOTE, D. H. (1967). Metabolic relationships of the isolated fractionsof the pectic substances of actively growing sycamore cells. Biociicm. J. 105, 45-59.

STREET, H. E., COLLIN, H. A., SHORT, K. & SIMPKINS, I. (1968). Hormonal control of celldivision and expansion in suspension cultures of Acer pseudoplatanus, L : the action of kinetin.In Biochemistry and Physiology of Plant Growth Substances (ed. F. Wightman & G. Setter-field), pp. 489-504. Ottawa: Runge Press.

SWAIN, T. & HILLIS, W. E. (1959). The phenolic constituents of Primus domestica. I. Thequantitative analysis of phenolic constituents. J. Sci. Fd Agric. io, 63-68.

TORREY, J. G. (1968). Hormonal control of cytodifferentiation in agar and cell suspensioncultures. In Biochemistry and Physiology of Plant Growth Sid>stances (ed. F. Wightman &G. Setterfield), pp. 843-855. Ottawa: Runge Press.

TORREY, J. G., FOSKET, D. E. & HEPLER, P. K. (1971). Xylem formation: A paradigm of cyto-differentiation in higher plants. Am. Scient. 59, 338-352.

TREVELYAN, W. E., PROCTER, D. P. & HARRISON, J. S. (1950). Detection of sugars on paperchromatograms. Nature, Lond. 166, 444-445.

VAMOS, L. (1953). Application of^>-aminophenol for the detection of sugars in paper chromato-graphy. Magyar kdm. Foly. 59, 253-254 (Chem. Abstr. (1954) 48, 506.)

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{Received 10 January 1972)

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0-

NAA, mg/l.Figs. 2-7. For legends see p. 332.

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Fig. 2. Callus formed on a hypocotyl explant 8 days after placing on solidified PRL 4with 1 mg/1. NAA and 2 % sucrose. The wedge of callus at the end of the explant isclearly visible. Tube diameter was 25 mm.Fig. 3. An excised hypocotyl segment similar to the one shown in Fig. 2. Roots arepresent on the explant. Tube diameter was 25 mm.Fig. 4. A section through S 4 callus showing the normal callus cells and cells whosevacuoles are filled with a dense amorphous material. Some cells containing this materialhave not embedded well. Glycol methacrylate section stained with picric aniline-blue.X320.

Fig. 5. A section through a hypocotyl forming a callus (see Fig. 2). The cortical cellshave expanded and broken away on the right of the picture and the meristematic cellshave proliferated to form a wedge. Wax section stained with picric aniline-blue, x 130.Fig. 6. Deposits on the inside of the tonoplast. The larger deposits were blue and thesmaller ones red when stained with safranin/picric aniline-blue. Wax embedded.X780.

Fig. 7. The effect of NAA on the fresh weight of S 4 callus. One of the replicates is usedto mark the points on the histogram. Actual size.

The roots shown in Figs. 8-13 were growing from callus incubated on PRL4 with1 mg/1. NAA, o-i mg/1. kinetin and 2% sucrose.

Fig. 8. Roots growing from the surface of S 4 callus. Tube diameter, 25 mm.Fig. 9. A 'bald' root approx. 3 mm long growing from the callus surface.Fig. 10. Two roots, approx. 4 mm long, which carried hairs along the whole of their

length.Fig. 11. A root which appears to have abruptly developed geotropic sensitivity. The

root hairs are unevenly distributed. Approx. x 5.Fig. 12. A cross-section through a root 4 mm long. The triarc pattern of the stele and

the root hairs arising from the epidermal cells can be seen, x 150.Fig. 13. A longitudinal section through the root shown in Fig. 12. The extent of

development of the root cap is visible, x 205.

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Figs. 14-18 show sections of callus grown on PRL4 with 1 mg/1. NAA, o-i mg/1.kinetin and 2 % sucrose; Figs. 19, 20 show sections of callus grown on a similarmedium but with 1 % sucrose. Figs. 15-20 show sections stained with dilute aniline-blue and viewed with ultraviolet light.

Fig. 14. A section of a well developed nodule. The central xylem with thickenedwalls, the small cambial cells and the cells filled with deposits can be seen. Methacrylatesection stained with picric aniline-blue, x 490.

Fig. 15. An extensive area of differentiation within a band of mitotic activity justbelow the callus surface is shown, x 200.

Fig. 16. A higher-magnification photograph of an area from the same slide as thatshown in Fig. 15. The phloem around the central xylem (arrows) can be seen, x 510.

Fig. 17. A section through a nodule with extensive xylem differentiation encircled byphloem. Serial sections showed that this nodule had developed into a root, x 200.

Fig. 18. A high-power photograph of the paired callose pads of a sieve plate, x 810.Fig. 19. In the section of this callus an area of xylem within a cambial area can be

seen, x 190.Fig. 20. A section showing an area of xylem with adjacent phloem (arrows), x 190.

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Fig. 21. The effect of simultaneously varying the kinetin and sucrose concentrations onthe growth and differentiation of S 4 after 8 weeks. Roots present on the calluses areindicated by arrows. Roots are absent on those calluses grown on media containing lessthan 2% sucrose. Callus diameter in the bottom 3 rows was approx. 25 mm.Fig. 22. These calluses were grown for 8 weeks on PRL 4 with 1 mg/l. NAA and variousadditions: a, 3 % raffinose; b, 3 % raffinose, 01 mg/l. kinetin. Roots are present; c, 3 %trehalose; d, 3 % trehalose, o-i mg/f. kinetin. A root is visible (arrow). Approximatesize of a, 25 mm long.

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Kinetin, mg/l.

005 0-1 0-2 0-3 0-5 10 20

22-2

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