On the Determination of Vascular Patterns During Tissue Differentiation in Excised Pea Roots Author(S): John G

Home , Vascular tissue

On the Determination of Vascular Patterns During Tissue Differentiation in Excised Pea Roots Author(s): John G. Torrey Source: American Journal of Botany, Vol. 42, No. 2 (Feb., 1955), pp. 183-198 Published by: Botanical Society of America Stable URL: http://www.jstor.org/stable/2438468 . Accessed: 23/08/2011 15:22

Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp

JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected].

Botanical Society of America is collaborating with JSTOR to digitize, preserve and extend access to American Journal of Botany.

http://www.jstor.org February,1955] TORREY-VASCULAR PATTERNS IN EXCISED ROOTS 183

throughout the United States. (3 vol. and atlas). POTONIE, H. 1889. Uber einige Carbonfarne. Jahrb. K. Second Geol. Surv. Pennsylvania Rept. Progress P. Preuss. Geol. Landesanst. 10: 21-27. p1. II-V. MAMAY,S. H. 1950. Some American Carboniferousfern RENAULT, B. 1883. Cours de botanique fossile. Vol. III. fructifications.Ann. Missouri Bot. Gard. 37: 409-476. Paris. MOORE, R. C., ET AL. (Pennsylvaniansubcommittee of the WILSON, C. L. 1953. The telome theory. Bot. Rev. 19: National Research Council committeon stratigraphy). 417-437. 1944. Correlationof Pennsylvanianformations of North ZIMMERMAN, W. 1930. Phylogenie der Pflanzen. Jena. America. Bull. Geol. Soc. America 55: 657-706, chart . 1952. Main results of the "Telome theory." The no. 6. Palaeobotanist 1 (Birbal Sahni mem. vol.) : 456-470.

ON THE DETERMINATIONOF VASCULARPATTERNS DURING TISSUE DIFFERENTIATION IN EXCISED PEA ROOTS' JohnG. Torrey

ONE OF THE mostcharacteristic and diagrammatic Biinning)have clearlydemonstrated the continuous patternsestablished in theseedling structure during early establishmentof the radial vascular pattern the ontogenyof the individualplant is the alternate in the root proximalto the apical initialsand the and radial arrangementof the primaryvascular uninterruptedacropetal maturation of vascular tissueswithin the primaryroot. The determination tissues. of a precise radial arrangementof alternating The experimentalstudies, limited in number, strandsof phloemand xylemtissue by longitudinal have been reviewedrecently by Esau (1954). Two differentiationoccurs during early embryogeny. major aspectsconcerning the determinationof the The vascular patternis evident in the seedling pattern of vascular differentiationhave been radicle. In many species,this radial patternwith analyzedusing experimentalmethods. Jost (1931- its characteristicnumber of vascular strandsmay 32) fromhis studieson vascular differentiationin' persistfor the life of the primaryroot. regeneratingdecapitated roots concludedthat the Prior investigationsof the originof the vascular maturevascular tissues of the root induce differ- patternin roots presenttwo general approaches, entiationof thenewly formed cells derivedfrom the each of whichmay have significancein understand- apical initials into the establishedpattern of the ing the basis of the pattern.One approachinvolves matureregion. This conceptof longitudinalinduc- detailedanatomical studies of the.root structurein tion arises from Pfeffer(1904) and Haberlandt all parts of the root system-primaryaxis as well (1913) 'who reportedthe controlof cell division as branchroots of differentsizes and ages. The and differentiationof meristematiccells by mature otherapproach has been one of experimentalmanip- differentiatedvascular tissues. Supportfor the idea ulation,usually by surgicaloperation, in an attempt of vascular tissue determinationby inductionwas to influencethe tissue differentiationand pattern givenby someearly root-culture work (e.g., Bonner formation. and Addicott,1937). Furtherindirect support for Descriptivestudies reportedextensively in the this view of differentiationhas been given by the classicalanatomical literature (van Tieghem,1870- consistentobservation in anatomicalstudies of the 71; DeBary, 1884) have been interpretedin such acropetal sequence of vascular tissue maturation works as Bower (1930) and are summarizedby in roots. Esau (1953). It has been shownthat the number of alternateand radiallyarranged xylem and phloem The opposedview holds that the determination of- strandsmay be constantin any givenplant and may the vascular patternin roots is controlledby the^ be quite characteristicof a species,genus, or even apical meristemwhich governsthe differentiatiorn family. In some plant groups the number of of the primarytissues of theroot. Thoday (1939)' vascularstrands in the primaryroot varies among has emphasizedthe self-determiningpower of the membersof the group (Wardlaw,1928) or varies root apex and has cited the evidencederived from within the root of the same individual (e.g., culturingisolated root tips in nutrientmedium. As as Heimsch,1949; Meyer, 1930). Lateral branches early 1931 Scheittererconcluded from experi- fromthe primaryroot axis oftenshow a reduced mentswith very small excised root tips which grew number of vascular strands. Recent anatomical and differentiatedin nutrientculture that the root analysesof rootontogeny (e.g., Esau, Heimsch,and meristemcontrols primary tissue differentiationin' the root. Recently,Biinning (1952) has reported' 1 Receivedfor publication June 15, 1954. thatif a 2-mm.root tip of Vicia faba is excisedand' This investigationwas supportedin partby research replacedon the shortenedstump of the same root,. grantnumber RG 2861 fromthe NationalInstitutes of it would forma union and grow,but the xylem' Health,Public Health Service. The writeris indebtedto Mr. MiltonAnken for his elementsof the two parts are not in line and only excellenttechnical assistance. laterbecome connected by differentiatingelements. 184 AMERICAN JOURNAL OF BOTANY [Vol. 42

He concludedthat the vascular pattern was already elsewhere(Torrey, 1954). The procedurecan be fixedin the 2-mm.root tip and thatdiffusible sub- summarizedas follows: Alaska pea root tips 5.0 stancesmoved fromthe maturetissues toward the mm. in lengthwere excised from sterilizedseed tip which made possible the maturationof the germinatedfor 48 hr. and weregrown in a synthetic vessel elements. nutrientmedium containing mineral salts, 4 per The secondmajor aspectof vasculartissue differ- cent sucrose and 0.5 per cent agar, plus FeCl3, lack- entiationin rootsconcerns the question of thedeter- ing added vitamins and trace elements. After minationof theradial and alternatearrangement of growth for 1 week to an average root length of xylem and phloem tissues in the primaryroot. about 50-60 mm.,the terminal0.5-mm. tips (includ- Accordingto thedescription of differentiationin the ing root cap) were excised from the control roots diarch roots of Sinapis by Buinning(1951), the under a dissecting microscope equipped with an early enlargementof the centralfuture-metaxylem ocular micrometer and were transferred to fresh elementsimmediately behind the apical initials medium in small dishes. Growth of these experi- exercisesa determininginfluence on the subsequent mental tips was dependent upon the addition of the differentiationof all the tissues of the central vitamins, thiamin and nicotinic acid, and trace cylinder.This influenceresults in the inductionof elements. Such 0.5-mm. root tips were grown for differentiationofthe radially-aligned vessel elements various periods from one to several weeks and were of the xylemand the suppressionof phloemtissue then killed in formalin-aceticacid-alcohol (FAA) except at the radial points most distantfrom the with aspiration. Root tips were dehydrated in an induced xylemtissue. Accordingto Biinning,no ethyl-butylalcohol series, embedded in "Tissue- phloemcan arise in the immediateneighborhood Mat" and sectioned on a rotary microtome at 8,u. of a xylemstrand nor in the vicinityof existing Serial sections were stained with Heidenhain's phloemtissue. Thus a radial and alternatepattern hematoxylin and safranin. Histological study of of vascular tissue must result. Biinning (1951, large numbers of roots in any one experimentwas 1952) has used stainingreactions, measurements of accomplished with roots killed in FAA and sectioned nucleolarsize, and the resultsof woundingexperi- at 40M using a carbon-dioxide freezing microtome. mentsto supporthis speculations. Sections were mounted in water and examined under In studyingthe determinationof vascular-tissue the microscope immediatelywithout staining. patternsin the root, it is essentialto analyze the The root diagrams used as illustrationswere made problemnot in termsof two-dimensionaltransverse by drawing cellular outlines directly on photo- or longitudinalroot sections,but in the light of graphic prints, removing the emlusion and com- the three-dimensionalroot structureas a whole, pleting the line drawings in ink. Both the original consideringlongitudinal as well as radial forces photomicrographand its line tracing are presented actingupon the system.The problemthen resolves to facilitateinterpretation. itselfinto two fundamentalquestions. First: is the Structure and vascular tissue differentiationin determinationof the acropetal differentiationof excised control pea roots in culture.-The organiza- primaryvascular tissues in theroot inducedby the tion of the meristematicregion of the root apex in older and maturevascular tissues or is such deter- the cultured pea roots studied appeared quite minationinherent in theactivity of a self-determininguniform. Figure 1 shows, in median longitudinal apical meristem?Second: whatis thenature of the view, the typical organization of the apical region forcesthat produce the radial and alternatearrange- of a pea root fixed at the start of the culture period. mentof the primaryvascular tissuesof the root? This organization is maintained in elongating roots Both the observationaland the experimental in culture and representsthe arrangementof tissues approacheshave been used in an attemptto find of the root apex in week-old roots before 0.5-mm. answers to these questions. The first of these tip excision. problemsis the subjectof the presentinvestigation. A group of apical initials gives rise to the cells A detailed anatomical analysis of the course of of the central cylinder by cell divisions which early primaryvascular tissue differentiation in untreated delimit the procambial cone proximal to the apical controlroots grown in nutrientculture has been initials. The same initials give rise to cells distally used as the backgroundfor the experiments.From which undergo transverse divisions, forming the anatomicalstudies of culturedroots, it was possible columella of the root cap. The cortical initials to determinethe properlength for excision of root appear to lie peripheral to the apical initials where tips in whichno maturevascular tissues occurred the cortex and epidermis have a common origin and in whichincipient vascular patternformation with the cells of the root cap lateral to the columella. had just begun. Such root tips were successfully Thus, although the central procambial cylinder has subculturedin a sterilesynthetic nutrient medium a clear-cut initial group, the other primary meri- and thecourse of vasculartissue differentiation was stematic tissues show no such precise site of origin studied. (fig. la). The relativelymassive subterminalapical MATERIALSAND METHODS.-Procedures forgrow- meristemin Pisum thus includes the poorly individ- ing 0.5-mm.pea-root tips in a sterile synthetic ualized apical initials, which give rise more or less nutrientmedium have been described in detail directly to the primary meristematictissues of the February,1955] TORREY-VASCULAR PATTERNS IN EXCISED ROOTS 185

ffi'14 I 7 s ~~~~~~~~~~~~~~ita

Fig. 1-3.-Fig. 1. Median longitudinalsection of root tip of Pisum sativum variety'Alaska,' excised from seed 48 hr. after germination.See photographictracing in fig. la for cellular detail. X 175.-Fig. 2. Transverse section of one.week- old cultured root, showing procambial cylinderat 300,uproximal to the apical initials or 500Atfrom the extremetip. See photographictracing in fig. 2a for cellular details. X350.-Fig. 3. Transversesection of week-oldroot grownfrom 0.5-mm. pea root tip. Section was cut at 288A proximal to the apical initials, showing the triarch arrangementof the enlarged metaxylemelements and the firstmature protophloemelements. X 175. root, and the actively dividing cells intimately type" whichhas since been similarlycriticized by associated with them. The organization of the root Reeve (1948) forthe pea-embryoradicle. apex is apparently intermediatebetween the classic The groundmeristem originates by divisionsof open or "Pisum type" described by Janczewski the corticalinitials peripheral to the apical initial (1874) and evident in such roots as Daucus (Esau, zone. The innermostlayer of cells of this tissue 1940) and the clearly delimited tiered arrangement, continuescell divisions. A precise ring of cells is as seen in the roots of tobacco (Esau, 1941) and produced,marking clearly the boundary between the most monocot roots. One mightinterpret the initial centralcylinder and the peripheraltissues of the region in the pea root as an invertedcup not unlike root.The groundmeristem increases in cell number that described for Fagus by Clowes (1950). Tiegs radiallyfrom about 6 cells to an average widthof (1913) has already pointed out the erroneous 10-11 (fig.4). This radial increaseoccurs through nature of Janczewski's interpretationof the "Pisum successivepericlinal divisions of the inner layer, 186 AMERICAN JOURNAL OF BOTANY [Vol. 42

Pericycle

/0\ rstimatu 3 4 0 Fi re protophloem Region of apical initials

Enlarged future

600

la 2a

600 340 Firstmature protophloem

Level of , 275 Level of excision excision 500 ~~~~~~~~~~~~~~~~~260 Division of ex c x_ \\ \ \ < ( 19-20) > / / / mother cell

400 \-o (10) - t t / 175 First metaxylem

\ \ \ ~--~ ~ / Ground / / enlargement \ \ \ / ~~~~~~~meristem/ Procambium / /

.300

\Y\ t( 13-15)>) / | 40 Concentric pattern 0 Apical initials

20 0 \ \ 6/t- / /Distance from Distance from 2 ~ *4) Apical Meristem Extreme tip i n in ju |

\ \ | / Root cap 100 Columella 225 p

4 0.~~~~~~~~ 4 0~~ 1 Fig. la, 2a, 4.-Fig. la. Photographictracing of fig. 1, showing cellular detail. XIIO.-Fig. 2a. Photographictracing of fig.2, showing cellular detail. X175.-Fig. 4. Diagrammatic representationof tissue arrangementin 0.5-mm.pea root tip fromwhich experimentalmaterial was grown,showing the spatial relationshipsin tissue differentiation.See text for details. ApproximatelyX175. February,1955] TORREY-VASCULAR PATTERNS IN EXCISED ROOTS 187 whichfinally becomes identifiable as the single-cell- having a terminalconical shape proximalto the layered pericycle. The patternof concentriccell initialregion, may properly be termedprocambium. layersis evidentin transversesection within 30-40u The firstevident change in theseprocambial cells of the regionof the apical initialsand is the first as seen in serial transversesections is the enlarge- visibletissue pattern proximal to the initialregion. ment of metaxylemelements one in each of the Establishmentof the origin of the endodermis at this three future xylem strands. The first manifest level was not possibleas no distinguishingfeatures enlargementof these cells is observedan average were observed. Even in the matureroot, the endo- distanceof about175ku proximal to theinitial region dermisshows no evidentcharacteristic structures. (fig. 4). At this level the procambialcylinder has In longitudinalsections, cortical thickening is seen alreadyincreased in diameterthrough the formation also to occur throughT-divisions (Clowes, 1950) of new procambialcells by longitudinaldivisions to in intermediateand outer layers of the ground an average of 19-20 cells. In each radial xylem meristem(fig. la). arm, the metaxylemcells which firstenlarge are While the organizationof the apical region in seen to lie inside the midwaypoint on the radius isolated pea roots grownin cultureis quite con- betweenthe outermostlayer of the centralcylinder sistent,the actual diameterof the region tends to and the root center. Usually two or three small diminishslightly during early growthin nutrient procambial cells lie betweenthem and the root medium. The diameter decrease is attributable center. Subsequent enlargement,of individual chieflyto a reductionin the numberof cell layers metaxylemelements occurs along a radial path in the groundmeristem which becomes stabilized at outwardfrom the firstenlarged cells toward the a width of approximately10-11 cell layers. All futurexylem pole. At about 275tufrom the initial averagedimensions and cell countsof controlroots' region threemetaxylem mother cells in each arm presentedin the text or in tabularform represent have enlargedin diameterand the futuretriarch measurementsmade on rootsfixed after one week's xylem patternis clearly indicated. The increase growthin nutrientmedium. in diameterof thesecells usuallyrepresents only a In fig. 4 are representeddiagrammatically the two-foldincrease over adjacent procambial cells spatial relationshipsobserved in the root apex of a and althoughclearly evident in transversesection typical week-oldcontrol root grown in nutrient (fig. 2, 2a), one finds difficultyin picking out medium. In table 1 are presenteddetailed measure- preciselysuch cells in longitudinalsection close to mentsof a series of roots,from which the average the apical initials. It has not been possiblein these dimensionsshown in fig.4 weretaken. roots to trace such early enlarged metaxylem The central cylinderthus originatesfrom the elementsin singlefile into the initialregion. apical initialsand is set offfrom the future cortical The central procambial cells remain small in zone by a cylinderof cells formingthe innerlayer diameterand parenchymatousin appearancefor a of the groundmeristem. At about 30-40tubehind considerabledistance proximal to the apical initials the apical initials, the diameter of the central (usuallygreater than 10 mm.in the controlroots). cylinder averages 13-15 procambial cells which These cells apparentlyplay no role in the deter- have been producedby longitudinaland transverse minationof the radial vascular pattern.At some divisions of the cells derived from the apical stage afterthe enlargedmetaxylem elements have initials. The central cylinder is comprised of completed their maturation,these central cells elongatecells, 3-4 timesgreater in length than width. undergochanges leading to theirmaturation, form- These cells are of uniformtransverse diameter, ing a solid centralcore of primaryxylem (Torrey, have large nuclei and theirdensely staining proto- 1951). The delayed maturationof these central plastsshow little vacuolation. The cells are typically elementsis a matterof considerableinterest and procambialin appearanceand the entirecylinder, worthyof furtherinvestigation. As referredto in

TABLE 1. Longitudinal dimensionsin the apical region of isolated pea roots grown for one week in a syntheticnutrient medium,showing the spatial relationshipsin the primaryvascular tissues during early differentiation.Values in ,u.

Root Cap FirstMetaxylem Sieve Tube First Mature FirstMature Root Number itfrom tip Enlargement ElementDivision Phloem Xylem ,I fromApical Initials 1 -0-288 160 208 288 5180 2 -0-280 152 208 288 7260 3 - 0-192 216 272 352 5770 4 -0-168 208 224 288 5 -0-304 112 408 6 -0-160 152 440 7 -0-168 224 336 Averages-0-225 175 260 340 >5000 188 AMERICAN JOURNAL OF BOTANY [Vol. 42 this paper, protoxylemelements are the first ing this final longitudinaldivision, the outermost matured primaryxylem elementswhich usually cell adj oining the pericycleenlarges slightly,be- occupya characteristiclocus at theperiphery of the comes vacuolated,and undergoesthe characteristic centralcylinder. The metaxylemis that portionof changes to a mature sieve tube element (Esau, the primaryxylem maturing after the protoxylem. 1950) within a distance of about 60-80g. The Early and late metaxylemare not distinguished, sistercell whichis approximatelythe same size as although,strictly speaking, the enlargedmetaxylem thesieve tube element in diameteras well as length, elementswhich form the characteristicradial pat- remainsas a flankingparenchymatous cell usually terncould be interpretedas "earlymetaxylem" and on the innerside of the sieve tube element.Several the later maturingcentral elements as "late meta- additionalsieve tube elementsform and maturein xylem." similarfashion in rapid successionon eitherside of The early blockingout of the xylempattern by the firstmature elements, adjacent to the pericycle. cell enlargementof theradially-arranged metaxylem The origin of sieve tube elementsby longitudinal mothercells is characteristicof mostof the dicoty- divisionof protophloemmother cells, after increase ledonous and monocotyledonousroots reportedin in the diameterof the central cylinderby cell detail. It is clear howeverthat in Pisum, unlike division and cell enlargementhas occurred,has most monocotroots, e.g., Allium (Mann, 1952), been describedin a numberof dicotyledonousroots Hordeum (Heimsch, 1951), etc., and the diarch (e.g., Nicotiana,Esau, 1941) as well as in monocot dicot roots studied,e.g., Nicotiana (Esau, 1941), roots (e.g., Hordeum,Heimsch, 1951). In Daucus Sinapis (Biinning,1951), etc., metaxylemenlarge- no such divisionof protophloemmother cells has mentdoes not occur immediatelyadjacent to the been observed(Esau, 1940). apical initials,but is delayed until some distance At the level of thesefinal cell divisionsthat give behindthe initialregion. As has been pointedout rise to the firstmature sieve tube elements,enlarge- by Esau (1940, 1941) theearly enlargement reflects mentof metaxylemmother cells has progressedto sluggishcell divisionof thesecells accompaniedby the pointthat two enlargedcells are clearlyevident relativelyrapid increasein cell volume. The sur- in each xylemarm. Thus, when the cells finally roundingprocambium maintains a more uniform comprisingthe matureprotophloem pattern have rate of cell divisionwith no evidentincrease in cell just been formedby cell divisions,the xylem pattern diameter. At this level, no markeddifferential in is alreadyclearly established (fig. 2, 2a). cell lengthis evidentamong the cells, and, in Pisum, As has been described in the root of Pisum vacuolationof the enlargedmetaxylem elements is (Torrey,1951, 1953), thematuration of protoxylem delayed for some distance (averaging 400-500fL) tissueproceeds much more slowlythan does proto- until elongation of the immature metaxylem phloemmaturation. In the materialstudied here, elementsbegins. Then smallisolated vacuoles form matureprotoxylem elements identifiable by lack of adjacent to the two end walls of the elementand nucleus and the presence of lignifiedsecondary eventuallycoalesce withinthe elongatevacuolated walls,were firstevident between 5000-6000,u (5-6 elements. mm.) behindthe apical initials. Xylemmaturation Midway betweenthe radial arms of the future occurs centripetallyand typicalexarch xylem is xylemand immediatelywithin the cylindricalperi- foundin the primarybody. In culturedpea roots, cycle cell layer lie the futurephloem areas. At an completionof primaryxylem maturationusually averagedistance of 340kufrom the apical initialsthe occurs at distancesgreater than 10 mm. fromthe firstmature protophloem elements are evidentin root tip. transversesection. In each of the three distinct The week-oldcultured roots grownunder con- phloemareas, single sieve tube elementsare seen, trolled conditions showed remarkablyconstant each lying immediatelyadjacent to cells of the tissue differentiationin terms of patternand of pericycle (fig. 2a, 4). Each mature elementis actual positional relationships(table 1). In the readilydiscernible by the lack of cell contents,the hundredsof control roots examinedin sectioned characteristicpentagonal or four-sidedshape and material,the triarch arrangement of vascular tissues thedark staining cell wall. The firstthree elements, was alwaysobserved. In the experimentsdescribed one at each pole, usually,mature at slightlydiffer- below, the startingexperimental plant materials ent levels, but within30-40/A of each other. In were 0.5-mm.root tips,including root cap, excised tracingthe origin of these firstprotophloem ele- fromthese one-week-oldroots. Figure 4 therefore mentstoward the apical initials,it was foundthat representsdiagrammatically the excised 0.5-mm. tip the file of cells whichfinally produces the mature used as the startingexperimental material. At this sieve tube elementsoriginates regularly by a longi- level of excision the triarchpattern of vascular tudinalcell divisionof a protophloemmother cell. tissues is well established. Enlargementof three This division occurs 60-80,u distal to the first metaxylemmother cells in each xylem arm has maturephloem elements or at an average level of occurred and the site of the futuresieve tube 260,tbehind the apical meristem.The longitudinal elementshas been fixed. Figures2 and 2a showthe divisions occur in radial, periclinal or oblique stage of differentiationof the procambialcylinder directionwithout any apparentregularity. Follow- at the excisionsurface. Usually no matureproto- February,1955] TORREY-VASCULAR PATTERNS IN EXCISED ROOTS 189 phloemwas includedin the excised tips, although TABLE 2. Summary of root materials studied histologically, consideringthe possibilityof variationdue to the showing the observed incidence of transitionsin vascu- excisionprocedure, certain tips may have included lar tissue patterns. the earliest mature protophloemelements. No maturexylem tissue was presentin theexcised tips. Roots grownfrom 0.5-mm. root tips No. of roots Vascular tissue differentiationin roots developing from 0.5-mm. root tips in culture.-Half-mm. pea Studied in serial paraffin sec- root tips excised from week-oldroots grown in tions: culturewill increase their initial length about 15-20 Triarch throughout-24 timesin a week. The rootsare whiteand healthy, Diarch throughout- 5 but somewhatsmaller in diameterthan the roots Monarch throughout- 2 fromwhich they were excised. During extensive Diarch to triarch- 3 Monarch to diarch- 2 experimentson the nutritionalrequirements of 0.5- Monarch to diarch to triarch 1 mm. tips, (Torrey, 1954), numeroussamples of rootsof differentlengths grown in differentnutrient 37 media were fixedin FAA, usually afteronly one Studied in freezing microtome weekin culture,and wereembedded in paraffinfor sections: detailedhistological examination or were sectioned Sectioned at base: witha freezingmicrotome. Nearly 200 rootswere Triarch -66 examinedhistologically. During these studies,in Diarch - 11 whicha numberof anatomicalabnormalities were discovered,no evidencesuggested any correlation 77 between the anatomical changes observed and Total 114 changesin the nutrientmedia in whichthe roots grew. Anatomicalmodifications appeared in roots % of roots from 0.5-mm. tips grown on many of the differentnutrient media, showing pattern transition 21% includingthe mediumthat produced a maximum Sectioned at tip only: Triarch - - 32 rate of root elongation.In describingthe structual differenceshere, the nutrientmedium differences Roots grownfrom one-mm. root tips have thereforebeen disregardedsince their effect Studied in freezing microtome concernedelongation rate chiefly.As will become sections: apparentin the subsequentdiscussion, the observed Triarch throughout 16 abnormalitiesin tissuepatterns appear to be attrib- utableto the excisionprocedure and to the size of Total number of roots studied 162 the excisedtip. A largeproportion of the 0.5-mm.root tips grew as normalroots with the usual triarcharrangement of the vasculartissue. In theseroots the sequence of vascular tissue differentiationat the meristem excision. At the point of excision, many cells were was quite comparableto that describedabove for crushed or torn open by the cutting operation and thecontrol roots from which the tips were derived. these were manifest in sectioned material for a The root diameteras well as the diameterof the distance of as much as one mm. distal to the cut. maturevascular cylinder was slightlyreduced, due In fig. 5, 5a is illustrated in transverse section the to a reduced number of cells in the total root central cylinder from the basal portion of a root, diameter(compare fig. 2 and 3). Approximately showing the distortion and tearing caused by the 20 per cent of the rootsstudied in detail,however, excision. An immature,slightly asymmetric diarch showedabnormalities. The moststriking abnormal- xylem pattern is evident with phloem tissue irregu- ity observedin elongatingroots derivedfrom 0.5- larly arranged in three separate areas. The central mm.tips was the change in vascularpattern. This cylinder was flattenedlaterally by the excision and fundamentalchange involved a reductionin the the original triarch vascular arrangement was numberof vascularstrands accompanied by a com- destroyed. In fig. 11, Ila is illustrated the mature plete readjustmentof the tissues of the central symmetricaldiarch condition seen in another root. cylinder. Althoughthe 0.5-mm.tips were always In fig. 7, 7a is illustrated in transverse section the excisedfrom triarch roots, many of thesetips grew central cylinder of a root in which the arrange- as diarchroots and a numberwere observed which ment of vascular tissue is typically monarch. The showed a typicallymonarch arrangementof the central strand of xylem elements is surrounded vasculartissues. Table 2 lists the numberof roots by phloem elements arranged in the shape of a studiedhistologically and thenature of thevascular horseshoe. patternsobserved. The diarch arrangement of the vascular tissue, The changein vascularpattern was evidentat the either completely normal and symmetrical or base of the root very close to the level of the slightly asymmetric,was found at the bases of 19 190 AMERICAN JOURNAL OF BOTANY [Vol.42

40~~~4

A;; A , C A

-J. ~ ~ ~

ii. S: t . : _.ivAi:ii;2 li-

Fig. 5-12.- Photomicrographsof transversesections of roots grownill nutrientmedium fr.omexcised 0.5-mm.pea root tips, cut at various levels to show differencesin vascular arrangement. Root numbersrefer to table 3. Reference sold be made to photographictracings of these figuresin fig.5a-12a. All X350. -Fig. S. Asymmetricaldiarch arrangementill root 14 cut at 1200,ufrom exrtreme base, showinglateral compressionand tearing at the protoxylempoints due to excisilon. -Fig. 6. Triarch vascular arrangementin root 14, cut at 2400,u distal to the level shown inlfig. 5. Fig. 7. M>onarchvas- cular arrangementin root 17 cut at 940),ufrm the extreme root base, showing the horseshoe arrangementof phloem around the single xylem strand.-Fig. 8. Same root 17 cut-at 1415,u distal to fig.7, showingessentially diarch arrangement with one xylemelement ill the third strand,-Fig. 9. Same root 17 cut at 4640/Adistal 4a fig. 8 and 920,ufrom the apical meristem,showing the symmetricaltriarch arrangementwith enlarged metaxylemelements containing nuclei-ig. 10. Monarch vascular arrangementin root 11 cut at 2510,u from extre'meroot base, showing the opposite arrangementof February,1955] TORREY-VASCULAR PATTERNS IN EXCISED ROOTS 191

a a~~~~~~~~~~~~~~~

Fig. 5a-12a.-Photographic tracings of sections shown in fig. 5-12. Mature xylem elements are designated by shading of the thick secondarywalls; mature sieve tube elements of the phloem are indicated by thickened black walls. Refer- ence should be made to correspondingphotomicrographs in fig. 5-12 for detailed descriptions and cellular detail. All X350.

xylem and phloem.-Fig. 11. Same root 11 cut at 1400 distal to fig. 10, showing symmetricaldiarch vascular arrangement.-Fig. 12. Diarch vascular arrangementin root 12 cut at 2900ttproximal to the apical initials, showingthe symmetrical diarch arrangementjust proximal to the level of transitionto the triarchpattern. Future 'xylemelements of the third xylem strand can be identifiedand are indicated in the photographictracing, fig. 12a, numbered to show the centrifugal sequence of their maturation. 192 AMERICAN JOURNAL OF BOTANY [Vol. 42

roots.Five rootswere observed in whichthe pattern diarchor monarchcondition, if allowedto growin of vascular tissues at the base could be termed culture,ultimately reverted to the triarchvascular monarch,containing a singlestrand of xylemtissue arrangementcharacteristic of the pea root! The with a localized area of phloemtissue. The tissue transitionsfrom one arrangementto another arrangementillustrated in fig. 7 is typicalof the occurred at various root lengths. Table 3 sum- monarchcondition such as is foundin the root of marizesdata fromten rootsand showsthe position Op,hioglossum(Bower, 1930) and in roots of along the lengthof the root at whichthe transition certainother lower plant forms, and is an arrange- occurred.The transitionper se fromone patternto mentvery rarely observed under natural conditions the otherwas completedusually in about 1 mm. of in the roots of dicotyledonousplants (Guttenberg, root length. In one root that was sectioned(root 1940). 17, table 3), the completetransition from monarch At the timeof excisionof 0.5-mm.root tips,the throughdiarch to triarchoccurred within a distance triarchpattern of vascular tissues is well estab- of 5.3 mm. lished in the root tip. If the tissuesin whichthis In fig.5, 5a and 6, 6a are illustratedtransverse patternis determinedare not destroyedduring sectionstaken from root 14 of table 3, showingthe excision, the root grows as a triarch root. If, diarch and triarcharrangement at the two differ- however,the excision damage is sufficientlyexten- ent levels of pattern.In fig.7, 7a, 8, 8a, and 9, 9a sive, the triarchpattern is destroyedor partially the sequencefrom monarch to triarchis illustrated destroyedand newpatterns are producedwithin the in sectionscut fromroot 17 of table 3. In fig. 8 potentialitiesof the residual procambial tissues. the transitionfrom diarch to triarchhas occurred, Thus diarch roots develop or in some roots,even although the diarch arrangementis still clearly the verysimple monarch arrangement is produced. evident. In severalcases diarch or monarchroots It is of some significancethat under these condi- were killed and fixed before the transitionhad tionsthe new patternsalways represent a reduction occurred (table 2). These roots were all quite in the numberof vascularstrands within the root. short. In several diarch roots,it was possible to No matterwhat vascular patternis differentiated,study the earlydelimitation of the vascularpattern the tissues of the entire centralcylinder finally behind the apical initials. Such differentiation adjust by theformation of a symmetricalstructure. occurredin the mannerdescribed for the control In the case of the diarch roots formed,one xylem triarchroots except that only two xylemstrands arm may be relatedin originto a strandblocked wereinitiated. It is certainfrom these observations out in thepre-existing triarch pattern of theexcised that the meristemitself passed throughan active tip. This relationshipis difficultto establishwith period producingdiarch root structurebefore the certainty.The opposite xylem arm of the sym- transitionto the triarchcondition was established metrical diarch arrangementcannot be related, in the meristem.In everyroot in which sufficient however,to eitherof the two otherstrands of a elongation had occurred, the transitionto the pre-existingtriarch pattern, as the new strandlies "geneticallyfixed" triarch arrangementof the in a position midway betweenthem. Thus the primaryvascular tissues was accomplished. patternformed in the apical meristemhas changed, The anatomyof thevascular transition region.- as is clearlyevident by a studyof the meristematic Anatomicalstudies were made of rootsin whichthe regionin a numberof such roots. transitionfrom the diarch to the triarcharrange- Transitionsin vascular patterns.-Diarch and menthad been completedshortly before the root monarchroots do not continueto grow in culture was killed. In such roots,the formation of thethird witha fixedvascular pattern as in culturedtriarch xylem strand could be studied during the early pea roots. All roots in which.a reductionhad stages of maturationof the newlyformed xylem. occurredin the numberof vascular strandsto the Since all the cells comprisingthe central area of

TABLE 3. The occurrenceand position of transitionsin vascular tissue patternsin roots grownfrom 0.5-mm. tips.

Distance of new pattern Lengthof from apical meristem Root Number Vascular pattern Root lengthin A Transitionin ,u in ,u 8 -Monarch throughout 3050 9 -Diarch throughout 4225 10 -Diarch throughout 5760 11 -Monarch to diarch 7040 1065 4515 12 -Diarch to triarch 6690 370 1970 13 -Diarch to triarch 7225 840 3985 14 -Diarch to triarch 7400 1185 4975 15 -Diarch to triarch >8880 520 16 -Diarch to triarch 7380 6430 17 -Monarch to diarch to triarch 7970 232, 960 6270, 920 February,1955] TORREY-VASCULAR PATTERNS IN EXCISED ROOTS 193 the central cylinder,including the diarch xylem maturationof the newlyformed xylem strand was elementsthemselves, are enlargedand vacuolated, observed. it was not possible to distinguishthe initiationof The change in vascular patternfrom the single the thirdxylem strand by differentialcell enlarge- strand to the diarch arrangementwas somewhat mentas is possiblein the apical regionof the root. less precise and thus more difficultto determine Even so, the sequence of xylemmaturation of the critically. In table 3, roots 11 and 17 were newlyformed xylem strand was readilydiscerned studiedin detail over the region of transition.In root by a studyof serial transversesections. The first 11, illustratedin fig. 10, 10a, the monarch conditionis representedby a indicationof thematuration of theadditional xylem one-sideddistribution of the phloemtissue with a centralstrand of xylem strandwas the appearance of lignifiedsecondary elements. The transitionto the diarch condition walls in one of the enlargedvacuolated cells. This involvedthe gradual appearanceof a diametrically cell, located on a radius extendingfrom about the opposedphloem tissue and thecentrifugal extension mid-pointof the diarch xylemplate, corresponds of the xylemband by differentiationof enlarged in position approximatelyto that of the earliest vacuolated cells to form the symmetricaldiarch enlargedmetaxylem elements in the alreadyestab- condition(fig. 11, la). In root 17, illustratedin lishedxylem strands. Thus the innerlargest xylem fig.7, 7a, in whichthe monarchcondition is mani- elementof the new strandwas the firstto mature. fest in a horse-shoedistribution of phloemtissue Subsequently,distal to the firstelement a second surroundinga strand of xylem elements,there enlargedcell externalto the firstmaturing element occurredfirst a reductionin the numberof xylem began to show secondary wall thickeningand elementsto three elementsin transversesection lignification. In a similar manner, additional (distal to the level illustratedin fig.7). This con- smallerelements in a radial rowoutward from these ditionwas followedby thecentrifugal differentiation firstmaturing elements also differentiatedas xylem of a secondstrand opposite the first xylem elements elements,forming a new mature primaryxylem with the ultimate establishmentof two xylem strand. The positionof the radial arm is such as strands at 1800 from each other, separated by to forman approximatelyequal tripartitedivision enlargedand vacuolatedcells ofthe central cylinder. of the centralcylinder with the ultimateestablish- Subsequentlya third xylem area appeared as ment of a symmetricaltriarch vascular arrange- described (fig. 8) and the full transitionfrom ment. The appearance of the thirdxylem strand monarchto triarchwas complete(fig. 9). resultsin littleor no changein the diameterof the The relationof rouotsize to vascularpattern.-As centralcylinder. The directionof maturationof earlyas 1877 DeBary stated,as a generalrule, that the xylem elementsin the new xylem arm was as thethickness of a rootdiminishes, the number of exactly opposite that normallyobserved, that is, its radial vascular strandsalso diminishes.Bower maturationwas centrifugalinstead of the usual (1930) more specificallystressed the relationship centripetalmaturation. In fig. 12 and fig. 12a is betweenthe diameterof themature central cylinder representedin transversesection a diarch root and the numberof vascular strandsin the root, immediatelyproximal to the site of initiationof the citing this relationshipas evidence favoringhis third xylemstrand. In the diagram of the root, "Size-Factor"hypothesis. According to Bower(1930, fig. 12a, the futurexylem elements are markedby p. ix), the "Size-Factor"is "that influencewhich stipplingand theircentrifugal sequence of matura- tends to secure by modificationof Form a due tionis indicated.The elementnumbered 1 matured levellingup of the proportionof surfaceto bulk as firstand elements2 and 3 maturedsubsequently the Size increases." In referenceto the root, he and in that sequence, forminga strand of three quoted Wardlaw (1928) who had shown that in matureelements. The xylemelements became dif- theroots of a wide selectionof fernsand monocoty- ferentiatedin the midst of many pnlargedand ledonous and dicotyledonousplants studied the vacuolated procambial cells which initiallywere numberof vascular strandsin the root increased indistinguishablefrom the adjacenttissues. In this in almostdirect proportion to the increasein stelar root the maturationof the smaller centralmeta- diameter. Similar observationson the roots of xylemelements was not yet complete.No peculiar many plants have been recordedand the correla- arrangementof the phloemtissue was evidentprior tion has been noted by many investigators.Such to the initiationof the additional xylem strand. observationshave been made usually on different Sieve tube elementswere uniformlydistributed rootsof differentsizes chosen at randomfrom one along the peripheryof thecentral cylinder between plant or fromdifferent plants. Several reportsof the diarchxylem poles. The divisionof thecylinder this correlationhave been based on studies of a intothree sections by the differentiationof the new singleroot axis ofchanging diameter. In theswollen xylemstrand resulted in a divisionof phloem tissues storageroot of Maranta,Meyer (1930) foundthat on eitherside of the strand. In fourdifferent roots the numberof vascular strandsincreased with in- (table 3, Root 12-15) in whichthe transitionfrom creasing total root diameterand then diminished thediarch to thetriarch vascular arrangement could again to the original number past the swollen be studied in detail, the centrifugaldirection of zone. He also reporteda reductionin the number 194 AMERICAN JOURNAL OF BOTANY [Vol. 42 of vascular strands accompanyingreduced root central cylinder diameter of a number of the diameterin roots of Hyaucinthusand Asphodelus. experimentalroots describedearlier, arranged ac- Jost (1931-1932) observed in seedling roots of cording to increasingvascular complexity.Each Zea strikingreductions in vascularstrand numbers measurementis representativeof the root at the which were related to decreased diameterof the level of matureprimary tissues of a givenpattern. centralcylinder of the root. Similar observations Measurementsof the centralcylinder include the have been made by otherauthors (e.g., Bouillenne, pericycle. Roots which showed transitionsare 1928; Aldrich-Blake,1930; Hatch and Doak, 1933; representedby measurementsof the maturetissues Guttenberg,1940; Preston, 1943; etc.). Dodel at each level of vascular pattern. It is readily (1872) describedin detailthe reduction of vascular apparentfrom these data thatno correlationexists strandnumber in lateral roots as comparedto the between total root diameter and the degree of primaryroot, in severalspecies of Phaseolus. complexityof themature vascular pattern. Although Although both Wardlaw (1928) and Bower in his analysisof the effectof size on vascularcom- (1930) attemptedto explain the observedcorre- plexityin the shoot, Bower correlatesincreasing lationbetween central cylinder diameter and vascu- vascularizationwith total diameter, it is interesting lar complexityof the matureroot in termsof "size to notethat, in referenceto the root,the onlyvalid and structure"relationships, such an attemptwas correlationestablished was an increase in the at besta descriptionand is of littledirect assistance numberof vascularstrands paralleling an increase in analyzingthe physiologicalbasis of the pattern. in diameterof the vascular cylinderbut not total More recentlyThoday (1939) has pointedout that root diameter. Althoughtable 4 shows a general the diameterof the root meristemat the level of trendof increasingvascular complexity, i.e., mon- patternformation is the significantdimension in arch to triarch,associated with an increasein the an analysis of the causation and not the final diameterof the maturevascular cylinder, even this diameterof the matureorgan. relationshipis not significantsince considerable It is interestingto note the observedchanges in overlap in size of each of the differenttypes of vascular patternin the presentexperiments from vascular arrangementoccurs. Furthermore,there the point of view of the size-structurerelationship. is no consistentrelationship between size and vas- According to Bower's "Size-Factor" hypothesis, cular arrangementwithin a givenroot. In root 11, one would expect to find that a change in the the mature diarch arrangementof the vascular numberof vascular strandsin the root is corre- cylinderis larger (94,J) than the maturemonarch lated with a change in total root diameter. Table condition(574). Similarlyin root12, the diameter 4 presentsmeasurements of totalroot diameter and increasesfrom 83/ (diarch) to 1174i (triarch).On the otherhand, in root 17, the reversesituation is TABLE 4. Measurementsof roots derived from0.5-mm. tips evidentwith a continuousreduction in the diameter and control root tips, showing the transversedimen- of themature vascular cylinder accompanied by an sions of the mature primary tissues. Roots are listed increasein the numberof vascular strands:mon- according to increasing vascular pattern complexity. arch-103k; diarch-69,a; triarch-624. Individual roots with patternchanges are listed at each Following the suggestionof Thoday (1939), patternlevel. analysisof the size-structurerelationship was made in roots at the level of the pattern im- Diameterof Total Root formation, Root Vascular Centralcylinder Diameter mediatelyproximal to the apical meristem.Table Number Pattern No. of cells Widthin in, 5 presents representativemeasurements of the diameterof the procambialcylinder of rootsat the Roots grownfrom half-mm. tips. level of the earliestmaturation of protophloemele- 11 Monarch 10 57 507 ments,approximately the level at whichthe vascular 8 Monarch 9 78 585 patternfirst becomes established. Fewer roots were 17 Monarch 12 103 780 available for measurementwhich showed the re- 10 Diarch 9 63 187 17 Diarch 11 69 390 duced vascularpattern at the meristemat the time 12 Diarch 15 83 296 of killing. It is evidentfrom this table that a 11 Diarch 12 94 296 muchclearer trend relates the increased procambial 14 Diarch 14 114 840 diametersto increasedvascular complexity.Two 9 Diarch 14 125 605 types of triarch roots are included- (1) roots 17 Triarch 12 62 304 derivedfrom 0.5-mm. excised root tips which de- 14 Triarch 15 89 324 veloped normallyand (2) controlroots, initially 18 Triarch 15 132 390 derived from 5-mm.tips from germinatingseed 12 Triarch 14 117 351 and grownfor one week withoutexcisioh. Roots Controlroots grownone week in culture from 0.5-mm. tips show consistentlysmaller vascu- 3 Triarch 19 172 390 lar cylinder diameters-both in actual width and in 1 Triarch 20 176 449 number of cells, compared to the control roots, 4 Triarch 20 187 488 which are remarkably uniform among themselves. 2 Triarch 23 195 525 It is apparent that the 0.5-mm. roots were consist- February,1955] TORREY-VASCULAR PATTERNS IN EXCISED ROOTS 195

TABLE 5. Measurementsof roots derived from0.5-mm. tips older mature vascular tissues, as maintainedby and fromcontrol root tips, showing the dimensionsof Jost. Half-mm.pea-root tips, in which the pre- the procambial cylinder at the level of pattern incep- existenttriarch pattern of the primaryvascular tion. Roots are arranged according to increasing vass tissueswas destroyedby experimentalmanipulation, cular complexityand increasing diameter. producednew and differentvascular patterns during Incipient Diameter of procambial subsequent root growth. These new patterns, Root Vascular cylinder typicallydiarch or monarch,were maintainedfor Number Pattern No. of cells Widthin,u varyingdistances during root elongation,and then the triarchpattern was restored. In these experi- Roots grownfrom 0.5-mm. tips mentsthere was no inductionof the monarchor 8 Monarch 9 109 diarchpattern in the newlyformed 10 Diarch 9 47 cellularderiva- 11 Diarch 10 78 tives of the meristemas would be expectedfrom 9 Diarch 14 82 Jost's inductiveforces. The presence of mature 14 Triarch 14 94 vascular tissues in organic attachmentis not 16 Triarch 14 101 essentialfor the continueddifferentiation of pro- 19 Triarch 14 105 cambialtissues in theseroots, nor does thereappear 20 Triarch 15 105 to exist any direct controllinginfluence of the 12 Triarch 15 117 mature tissues over differentiationof the meri- 17 Triarch 16 117 stematic tissues. As suggested earlier (Torrey, 21 Triarch 14 140 1954), the mature differentiatedtissues of the 18 Triarch 17 146 normalseedling root may be thoughtof as providing Controlroots grownone week in culture and/or transportingessential nutritionalrequire- 3 Triarch 20 171 mentsthat regulate the activityof the apical meri- 4 Triarch 20 172 stem. When isolated and provided with a com- 1 Triarch 21 183 plete syntheticnutrient 2 Triarch 22 195 medium,the apical meri- 5 Triarch 22 195 stem with its immediatecellular derivativesap- 6 Triarch 23 195 pears to be able to developindependently. Thus, as has been suggestedfor the apical meristemof the shootby the workof Wardlaw (1947b, 1950) an(l ently smaller, incident to the excision. Even so, Ball (1948, 1950), the isolated apical meristemof many such roots were triarch (fig. 3). the pea root grown in nutrientmedium appears In comparing the procambial diameters in table to be self-determining,capable of producingits 5 with the diameters of the mature vascular cylinder own pattern and structurewithin the genetic of the same roots given in table 4 it is interesting capacities of the species. One must look, not to to note that no consistentrelationship exists between the maturetissues, but to the apical meristemfor these two dimensions in this particular experimental the physiologicalcontrol of the vascular tissue material. Thus one must not assume, as has been patternin such roots.2 done in the-past, that, in either experimentalor in It is evidentfrom these experiments with 0.5-mm. untreated material used for anatomical studies, a tips thatthe preformedpattern of vascular tissues constant relationship does exist between these at the apical regionwas destroyedby the excision dimensions that makes possible an analysis of the itselfin those roots whichproduced abnormal ar- differentiation process based on study of the rangements.In theseroots, damage and destruction mature vascular system alone. ofindividual cells ofthe future vascular tissue inter- The data in table 5 tend to confirm Thoday's ruptedthe course of their differentiationand re- contention (1939) that the number of vascular sultedin the differentiationof a reducedvascular strands in a root is related to the diameter (or system. In the roots showinghorizontal compres- circumference) of the central cylinder of the root sion at the base of the rootdue to the excision,the at the time of initiation-i.e., in the region of the suppressionof differentiationof one or more arms apical meristem,and not to the ultimate size of the of vasculartissue was evident(fig. 5a). Thus either mature root. Recently,-Wardlaw (1947a) has ad- diarchor monarchroots were formed where exten- mitted the improbability of any causal relationship sive destructionof the preformedpattern had oc- between the final mature functional condition and curred. In all cases, these new patternsresulted the initial processes of differentiation,and, in re- fromexcision and internaladjustment or rearrange- lation to the shoot at least, has concluded that the mature structure is related directly to the activity 2 In an article published since the above was written,E. of the apical meristem. Reinhard (Zeitschr. f. Bot. 42: 353-376. 1954) states this same general conclusion, based upon observationson the DIsCUSSION.-From the experiments described differentiationof tiny fragmentsof pea root tips culturedin above evidence suggests that the primary vascular sterile nutrientmedium. He concludes (p. 375), "Die nor- tissue pattern in the excised pea roots arises as a male Ausgestaltungder Wurzelstrukturist also wohl nicht product of abhaingigvon determinierendenEinfluissen iilterer Teile der the activity of the apical meristem and Wurzel, sondern von der Organisation des Vegetations- does not result from the inductive influencesof the punktes selbst." 196 AMERICAN JOURNAL OF BOTANY [Vol. 42 mentending in an essentiallysymmetrical arrange- pany increasingsize of the apical meristemand its ment. Boodle (1899) suggestedthat the monarch immediateprocambial derivatives. Such an expla- arrangementin the root of Ophioglossumwas nation, while clearly contributingto our under- derivedfrom the diarch condition in whichone xylem standingof the size-structurerelationship, in effect group had aborted, a view held earlier by van restatesthe problemof patternformation in roots Tieghem (1870-71). No evidenceconcerning the in termsof unitpattern determination through cellu- cause forthe natural suppression of the xylem group lar differentiation.In studyingnormal differentia- was offered.In the experimentsreported here, the tion at the apical meristemand the appearanceof excisionprocedure effectively reduced the diameter the vasculartissue pattern in roots of Pisum, it is manifestdue of theprocambial cylinder at thelevel at whichpat- clear thatpattern formation becomes enlargement. ternformation occurred and in manyroots the re- to differentialrates of cell divisionand of the futurepri- duced vascular patternresulted. It is significant That is, more rapid enlargement the metaxylemele- thatexcision of roottips at 1 mm.from the extreme maryxylem elements, especially to the of the pro- at a level wherethe vascular patternis well ments,occurs as compared rest tip, more producedno abnormalitiesof vascular cambialelements, which maintain a relatively established, cell division. Pattern differentiation(see table 2). rapid rate of longitudinal formationthen appears to be initiallydependent Reductionof the complexityof tissue arrange- upon forceswhich control, either directly or indi- mentsin thevascular cylinder in rootsof monocots rectly,cell division and cell enlargementin the and dicots by experimentalmeans has been de- apical meristem.The fundamentalproblem which scribedby several authors,using a varietyof ex- thepresent work raises concerns the nature of these perimental manipulations. Flaskamper (1910) forces and the means wherebytheir influenceis foundthat removal of thecotyledons from seedlings modifiedaccording to the dimensionsof the apical of Vicia faba resultedin retardedroot growth and a meristemand its derivatives.For the ultimateun- reductionin the numberof vascularstrands in the derstandingof thephysical-biochemical basis ofvas- root from6 to 4. Jost (1931-32), in his decapita- cular patternin growingroots, one muststudy and tion experimentson rootsof Zea, foundthat regen- analyzethe activitiesof the individualcellular de- erated tips producedfewer vascular strands,with rivativesof the apical meristemand the inter-rela- reductionsfrom 16 or 17 strandsto 10. Similarly, tionshipsexisting among them which determine the in regeneratingroot tips of Vicia seedlings,he ob- finalstructure. servedreduction of vascular strandsfrom 6 to 5, or from5 to 4. Fourcroy(1938) foundthat a trans- SUMMARY versepuncture of the roottip of Faba vulgarisand Isolatedpea-root tips cultured one weekin sterile Lupinus albus preventednormal differentiationof nutrientmedium show a uniformpattern of vascular vascular strandson the injured side of the root, tissue differentiation.The earliestvisible pattern with a progressivereturn to the normalcondition of primaryvascular tissue differentiation following distalto thewound. In similarexperiments in which the delimitationof the cylindricalprocambium is he cut wedgesin the regionof the apical meristem apparentin the enlargementof futuremetaxylem of Vickafaba roots,Clowes (1953) reporteda re- elements,beginning about 175[u proximal to the ductionin the numberof protoxylemarms after apical initialsand resultingin the blockingout of the excisions. He attributedthese changes to a de- the triarchprimary xylem pattern. The divisions crease in the size of the meristemproduced by the of the protophloemmother cells situatedon alter- incision. In all thesecases, as in the experiments nate radii occur at about 260u proximal to the reportedhere, the generalrule applies thatthe re- apical initials and mature sieve tube elements are ductionin the diameterof the centralcylinder of apparent within 340u of the apical meristem. Half- the root tip at the level of patternformation is ac- mm. root tips including root cap, which contain no companiedby a reductionin thenumber of vascular mature vascular tissues, were excised, grown in strands.In rootsof monocotsespecially, the change sterile nutrientmedium and analyzed histologically. in vascular patternmay involvestriking reduction Approximately 20 per cent of the tips studied of vascularstrands both peripheral and central,and showed a reduction from the normal triarch radial may not,in fact,be a situationexactly comparable pattern to a symmetrical diarch or monarch ar- to thatin typicaldicot roots. rangementof the vascular tissues. The reduced vas- In his analysisof root structure,Thoday (1939) cular pattern was evident at the root base and per- has suggestedthat the unitpattern in root vascular sisted during root elongation for varying lengths. tissue differentiation,i.e., the unitary vascular The abnormal pattern resulted apparently from a strandconsisting of xylemand phloem,may vary partial or complete destruction of the pre-existing withinrelatively narrow limits at the site of pat- triarchpattern in the tissues at the level of excision. tern inceptionand that the numberof repetitions After continued elongation, roots with a symmetri- of the unit patternmay thereforeincrease as the cal monarch or diarch vascular patternunderwent a dimensionsof the procambial cylinder increase. transition,returning to the original triarch arrange- Thus, increasingvascular complexity would accom- ment. This transitionusually occurred withinabout February,1955] TORREY-VASCULAR PATTERNS IN EXCISED ROOTS 197 one mm. of root lengthand involvedthe appear- notresult from inductive influences of theolder ma- ance of an additionalxylem strand by centrifugal ture vascular tissues upon the newlyformed cells differentiationto formthe new symmetricalvascu- producedby the apical meristemas was maintained lar tissue arrangement.Roots showingtransitions by Jost (1931-32) but arises as a productof the frommonarch to diarch, fromdiarch to triarch, activityof the apical meristem.Second, the deter- and, in one case, frommonarch through diarch to minationof the radial arrangementof the primary triarch,were studied.No correlationwas foundbe- vasculartissues appears to be relatedto the dimen- tween vascular complexityand either total root sions of the apical meristemat the timeof pattern diameteror the diameterof the maturevascular inception.It is suggestedthat pattern determination cylinder. A relationshipbetween the diameterof is underthe controlof unknownfactors which in- the procambiumat the level of patterninception fluencecellular division and enlargementin the and thecomplexity of thevascular pattern was sug- immediatederivatives of the apical meristem. gested. Two major conclusionswere reachedfrom DEPARTMENT OF BOTANY, theseobservations. First, the primaryvascular tis- UNIVERSITY OF CALIFORNIA, sue patternin 0.5-mm.excised pea root tips does BERKELEY 4, CALIFORNIA

LITERATURE CITED

ALDRICH-BLAKE, R. N. 1930. The plasticityof the root sys- auisseren Faktoren nebst einigen Bemerkungen fiber tem of Corsican pine in early life. Oxford Forestry die angebliche Heterorhiziebei Dikotylen. Flora 101: Mem. Clarendon Press. Oxford. 181-219. BALL, E. 1948. Differentiationin the primary shoots of FOURCROY, MADELEINE. 1938. Influence des divers trauma- Lupinus albus L. and of Tropaeolum ma/us L. Symp. tismes sur la structuredes organes vegetaux a evolu- Soc. Exp. Biol. II. Growthin relation to differentiation tion vasculaire complete. Ann. Sci. Nat. Bot. Ser. X. and morphogenesis. Cambridge Univ. Press. Cam- 20: 1-239. bridge,England. GUTTENBERG,H. VON. 1940. Der primare Bau der Angio- . 1950. Isolation, removal,and attemptedtransplants spermenwurzel. In Linsbauer's Handbuch 8; 3 Abt., of the central portion of the shoot apex of Lupinus 3 Teil. Berlin. albus L. Amer. Jour. Bot. 37: 117-136. HABERLANDT, G. 1913. Zur Physiologie der Zellteilung. BONNER, J., AND F. ADDICOTT. 1937. Cultivationin vitroof Sitzungsber.Akad. Wiss. Berlin 16: 318-345. excised pea roots. Bot. Gaz. 99: 144-170. HATCH, A. B., AND K. D. DOAK. 1933. Mycorrhizaland BOODLE, L. A. 1899. On some points in the anatomyof the other featuresof the root systemof pine. Jour. Arnold Ophioglosseae. Ann. Bot. 13: 377-394. Arboretum14: 85-98. BOUILLENNE, R. 1928. Anatomical material for the study HEIMSCH, C. 1949. Ontogeneticchanges in vascular tissues of growth differentiationin higher plants. Plant of maize roots. (Abstract). Amer. Jour. Bot. 36: 797. Physiol. 3: 459-471. . 1951. Development of vascular tissues in barley BOWER, F. 0. 1930. Size and form in plants. Macmillan roots. Amer. Jour. Bot. 38: 523-537. and Co. London. JANCZEWSKI, E. 1874. Recherches sur l'accroissementter. BUNNING, E. 1951. Ober die Differenzierungsvorgingein minal des racines, dans les Phanerogames. Ann. Sci. der Cruciferenwurzeln.Planta 39: 126-153. Nat. Bot. S6r. V. 20: 162-201. -. 1952. Weitere Untersuchungenuiber die Differ- JOST, L. 1931-1932. Die Determinierungder Wurzelstruk- enzierungsvorgiingein Wurzeln. Zeitschr. Bot. 40: tur. Zeitschr.Bot. 25: 481-522. 385-406. MANN, L. K. 1952. Anatomyof the garlic bulb and factors CLOWES, F. A. L. 1950. Root apical meristemsof Fagus affectingbulb development. Hilgardia 21: 195-251. sylvatica. New Phytol. 49: 248-268. MEYER, F. J. 1930. Die Leitbiindel der Radices filipendulae . 1953. The cytogenerativecentre in rootswith broad (Wurzelanschwellungen) von Maranta Kerchoveana columellas. New Phytol. 52: 48-57. Morr. Ber. Deutsch. Bot. Ges. 48: 51-57. DEBARY, A. 1884. Comparative anatomy of the vegetative PFEFFER, W. 1904. Pflanzenphysiologie.II. Aufl. Verlag organs of the Phanerogamsand ferns. Clarendon Press. von W. Engelmann. Leipzig. Oxford. PRESTON, R. J., JR. 1943. Anatomical studies of the roots DODEL, A. 1872. Der Uebergang des Dikotyledonen-Stengels of juvenile lodgepole pine. 'Bot. Gaz. 104: 443-448. in die Pfahl-Wurzel. Jahrb. Wiss. Bot. 8: 149-193. REEVE, R. M. 1948. Late embryogenyand histogenesis in ESAU, KATHERINE. 1940. Developmental anatomy of the Pisum. Amer. Jour. Bot. 35: 591-602. fleshystorage organ of Daucus carota. Hilgardia 13: SCHEITTERER, HERTHA. 1931. Versuche zur Kultur von 176-226. Pflanzengeweben. Arch. Exp. Zellforsch. 12: 141-176. . 1941. Phloem anatomy of tobacco affectedwith THODAY, D. 1939. The interpretationof plant structure. curly top and mosaic. Hilgardia 13: 437-490. Rept. BritishAssoc. Adv. Sci. 1: 84-104. . 1950. Development and structureof the phloem TIEGS, E. 1913. Beitrage zur Kenntnis der Entstehungund tissue. II. Bot. Rev. 16: 67-114. des Wachstums der Wurzelhaube einiger Leguminosen. . 1953. Plant anatomy. John Wiley and Sons, Inc. Jahrb. Wiss. Bot. 52: 622446. New York. TORREY, J. G. 1951. Cambial formationin isolated pea roots . 1954. Primary vascular differentiationin plants. following decapitation. Amer. Jour. Bot. 38: 596-604. Biol. Rev. 29: 46-86. . 1953. The effectof certain metabolic inhibitorson FLASKXMPER, P. 1910. Untersuchungen fiber die Ab- vascular tissue differentiationin isolated pea roots. hangigkeit der Gefiss- und Sklerenchymbildungvon Amer. Jour. Bot. 40: 525-533. 198 AMERICAN JOURNAL OF BOTANY [Vol.42

. 1954. The role of vitamins and micronutrientele- . 1947a. Experimental and analytical studies of ments in the nutritionof the apical meristemof pea Pteridophytes.X. The size-structurecorrelation in the roots. Plant Physiol. 29: 279-287. Filicinean vascular system. Ann. Bot. N.S. 11: 203- 217. VAN TIEGHEM, P. 1870-71. Recherches sur la symetriede . 1947b. Experimental investigationson the shoot structuredans les Plantes vasculaires. I. La racine. apex of Dryopterisaristata Druce. Phil. Trans. Roy. Ann. Sci. Nat. Bot. Ser. V. 13: 5-314. Soc. London B. 232: 343-384. WARDLAW, C. W. 1928. Size in relation to internal mor- . 1950. The comparativeinvestigations of apices of phology. 3. The vascular systemof roots. Phil. Trans. vascular plants by experimentalmethods. Phil. Trans. Roy. Soc. Edinb. 56: 19-55. Roy. Soc. London B. 234: 583-604.

PHYSIOLOGY AND ANATOMICALDEVELOPMENT OF TOMATO FRUIT TUMOR1 MichaelTreshow2

TOMATO FRUIT TUMOR, a disorder of green tomato of the fruitsat the timeof rubbing,and 6, 12, 18, fruitsobserved in Californiatomato fields and pack- 24, 36, 48, 72, and 96 hr. later. The tissue was ing houses, resultsfrom the rubbingencountered placed immediatelyin formalin-aceto-alcoholor a during picking,handling, and processingof the chromo-aceticfixing and killingsolution. Paraffin greenfruits. embeddedtissue was sectionedto a thicknessof Althoughtomato fruit tumor, sometimes referred 12[uand stainedwith safranin 0 and fast greenas to as waxyblister or oedema,was reportedas early describedby Johansen(1940). as 1896 by Selby (1896), no detailed studyhas There was no morphologicaldamage to the been made concerningthe anatomy,physiology, or epidermisor any of the underlyingtissue immedi- developmentof the disorder. ately after rubbing (fig. 1). The cuticle was Gardner(1925) made a studyof the disorder and usually intact, although occasionally the outer showedthat the tumorsresulted from cushions of portionof cutin was lacerated. hyperplastictissue which pushed up beneath the The firstsign of any abnormalityof the fruit necroticepidermis. The directstimulus was thought surfacewas the depositof tanninin the sub-epider- to emanate fromthe necrotictissue and initiate mal collenchyma,and sometimesin the epidermis, hypertrophyand hyperplasiain theunderlying cells. as earlyas 12 hr. afterrubbing (fig. 2). Six to 12 The firstsign of tomatofruit tumor is an incon- hr. later, the collenchymacollapsed and tannins spicuous wrinklingof the affectedarea whichwill weremore abundant.Within 24 hr. afterrubbing, later formthe tumor. Tumors are initiatedonly the outer layers of parenchymaor inner collen- on greenfruits and becomeless conspicuousas the chyma cells startedto enlarge under the rubbed fruitripens. They are formedonly from the affected area (fig. 2). These cells enlarged by intrusive area, or part of it, and do not spread laterally, growthand by crushingthe tannin-filledcollen- althoughthey may attain a height of 1-2 mm. chymatissue. Intrusivegrowth was mostprevalent Individualcircular or irregularlyshaped tumors are in the outer three or four layers of parenchyma generallyless than 1 cm. in diameteron fruitsin tissue. Some intrusivegrowth was evidentseveral the fieldand affectonly a smallportion of thefruit cell layers deeperin normaltissue, but it was not surface. Tumorswhich result from picking or pro- so extensiveas in the tumoroustissue. cessingare frequently1-3 cm. in diamneter.A large portionof the pericarp may be affectedby such Radial cell division may start in some of the tumors. parenchymacells within48 hr. afterrubbing (fig. ANATOMICALDEVELOPMENT.-Histological studies 3). Transversedivisions generallyfollowed (fig. were made of the developmentof tomato fruit 4), but were never so extensiveas the radial divisions.This tumor.Green Pearson tomato fruits, free of tumors, earlyhyperplasia could occurin the wererubbed with a roughslat froma packingcrate outermostlayer of parenchymatissue, or the inner- to stimulatetumor formation. All anatomicaland most collenchymatissue. In the lattercase, only physiological work Was conducted with fruits the collenchymacells next to the epidermiswere detachedfrom the plants. Sections of pericarptissue crushedand filledwith tannins. were removedfrom the shoulderand blossomends Nucleiof healthyand tumoroustissue did notdif- ferconsistently. However, nuclear divisions of tumor 1 Received for publication August 26, 1954.- A portionof cells wereoccasionally abnormal and occurredwith- a thesis submitted to the Graduate School, Universityof out cell wall formationso that a binucleatecell California,in partial fulfillmentof the requirementsfor the was formed.Other tumor cell nucleiwere Doctor of Philosophy degree. observed 2 The author is indebted to Dr. JohnT. Middleton forhis to be in a tetraploidcondition as determinedby advice and suggestions on this work. chromosomecounts.