The Development of Isolated of peregrina () in Culture Author(s): Patricia L. Goforth and John G. Torrey Source: American Journal of , Vol. 64, No. 4 (Apr., 1977), pp. 476-482 Published by: Botanical Society of America Stable URL: http://www.jstor.org/stable/2441778 . Accessed: 23/08/2011 16:13

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 Amer. J. Bot. 64(4): 476-482. 1977.

THE DEVELOPMENT OF ISOLATED ROOTS OF (MYRICACEAE) IN CULTURE'

PATRICIA L. GOFORTH AND JOHN G. TORREY Cabot Foundation,Harvard University, Petersham, Massachusetts 01366

AB STRA CT Seedlingroots of the sweetfern Comptonia peregrina (L.) Coult. were excised aseptically and culturedin a modifiedBonner-Devirian liquid nutrientmedium. elongationwas very slow in the basic mediumwhich containedinorganic salts, B-vitamins,trace elementsand 4%o sucrose. The additionof planthormones including gibberellic acid, indoleaceticacid, and zeatin,alone or in combinations,had littleeffect on growth.Myoinositol at 10 or 100 ppm doubled the rate of elongation.The effectof this sugar alcohol could not be replaced by scyllitol,D-sorbitol, D-mannitol or by increasingthe sucroseconcentration. Subcultured root tips showedprogressively less elongationin successivetransfers. Secondary thickening of the roots,especially in the basal half,occurred in initialpassages and in subculturedroots without added hormones.Root buds also occurredspontaneously especially in the basal portionsof culturedroots, both in firstand in successivepassages. An anatomicalanalysis showed that these buds were endogenous,arising from a secondarycortex of pericyclicorigin.

ISOLATED ROOTS ofherbaceous species growing in effortto establishisolated roots of Comptoniain sterilenutrient culture solutions have providedex- sterilenutrient culture. perimentalsystems for the studyof various prob- lems includingroot nutrition,lateral root and bud MATERIALSAND METHODS-Collectionand ger- formation,secondary thickening and nodule de- minationof fruitsof Comptonia followedproce- velopment (Torrey, 1965). Althoughestablish- duresreported by Del Trediciand Torrey(1976). ing roots of woody in culturehas been a Root tipsused to initiatecultures were takenfrom difficult process, limited success has been seedlingswhich had been startedfrom seeds ger- achievedwith the following plants: Acacia melan- minated on a sterileagar nutrientmedium con- oxylon R. Br. (Bonner, 1942), Robinia pseudo- taining10 ppm gibberellicacid (GA3). Whenthe acacia L. (Seeliger, 1956), Pinus spp. (Slankis, radicle of the germinatedseed had reached 2-3 1947; Barnes and Naylor 1959; Ulrich, 1962), cm in length,the terminalone-centimeter of the Acer rubrumL. (Bachelard and Stowe, 1963) radicle was excised aseptically,and transferredto and Picea abies L. (Momot et al., 1974). 50 ml of liquid-modified Bonner-Devirian(BD) Comptoniaperegrina (L.) Coult. (Myricaceae) medium (Torrey, 1956) in a 125-ml erlenmeyer is a woody shrubcommonly found in easternand flask. The modifiedBD medium contained the centralNorth America. The rootsystem of Comp- followingcomponents (in mg/l of glass-distilled tonia consistsof a taproot,numerous lateral roots water): 242 Ca(NO3)2 4H20, 42 MgSO4 . 7H20, and a fewwide-ranging and specializedhorizontal 85 KNO3, 61 KCI, 20 KH2PO4, 2.5 FeCl3 6H20, roots from which sprouts are readily formed. 0.1 thiaminHCI, 0.5 nicotinicacid, 0.5 pyridoxine Comptoniapropagates itself vegetatively by these HCI, 1.5 H3B03, 1.5 ZnSO4 7H20, 4.5 MnSO4 root sprouts, forminglarge clonal populations. H20, 0.25 NaMoO4, 0.04 CuS04 . 5H20, 40,000 Infectionof the root by a soil actinomycete-likesucrose, with the pH adjustedto 5.5 beforeauto- organismresults in the formationof root nodules claving. Any furtheradditions were made after capable of fixingatmospheric nitrogen (Ziegler and cold sterilizationof the solutionwith a Millipore Hiiser, 1963). Because of interestin the process filter. The root.cultures were incubated in the of nodule formationin these roots, we made an dark at 25 C. The elongationof the main axis of each root was measured and recorded weekly. ' Received for publication25 August 1976; revision At the end of a 6-8-wk period of cultureone- accepted8 November1976. centimetertips of the activelygrowing roots were The authorsare indebtedto Peter Del Tredici for excised and subcultured.The bases of the actively providingseeds and seedlingsof Comptonia,to Kathy growingroots were then transferredto a growth Kamo for assistancein culturingroots, to Monica Matt- mullerfor instructionin histologicaland photographic chamberwith 12 hr of lightand 12 hr of dark at techniques.The researchwas supportedin part by re- 25 C. Mixed whiteand fluorescentlights with a search grantBMS 74-20563 fromthe National Science total intensityof about 260 ft-cwere used. Foundation and in part by the Maria Moors Cabot Foundationfor Botanical Researchof Harvard Univer- Material for anatomicalstudy was handled by sity. standardparaffin methods; the roots were fixed 476 April, 1977] GOFORTH AND TORREY-CULTURE OF ROOTS IN COMPTONIA 477

8 II

7 10_ A

6 9 -

8 0 Z~~~~~~ *

35- 7 - A 'p3~~~~~~~~~~~~~~~~~1 6

2 A 5 -

4-. 4 * 0

oI I I I I . J ~AO C 2 3 4 5 6 Time (weeks) & o ~~V Fig. 1. The effectof various hormoneson the rate 24 L V v of elongationof isolatedroots of Comptonia. *-BD 0~~~~~ withno addition;A-10 ppm GA2; V-1.0 ppm IAA; 0-0.1 ppm IAA; A-1.0 ppm zeatin; 0-10.1 ppm zeatin. I I I I I O. , 1 2 . 3 4 5 G in formalin-acetic acid alcohol, dehydrated Time (weeks) througha tertiary-butylalcohol series,embedded in Tissueprep,and sectionedat 10 ,Am.The sec- Fig. 2. The effectof variousconcentrations of myo- tions were stained with Delafield's hematoxylin inositolon the rate of elongationof isolated roots of and safranin. Comptonia. Hexagon-BD with no additions; 0-1,000 ppm myoinositol;A-500 ppm myoinositol;0-100 RESULTS-The effectsof growth sub- ppm myoinositol;A-10 ppm myoinositol;*-1.0 ppm stances on root elongation-Excised root tips of myoinositol;V-0.1 ppm myoinositol. Comptoniagrown in BD mediumwith no added growthsubstances showed insignificantmain-axis elongation,i.e., approximately4 cm in 8 wk. that of Convolvulus arvensis L. at 20 mm/day Tests weremade of a numberof plantgrowth sub- (Bonnett and Torrey, 1965). As the roots ma- stances known to be importantin root develop- tured they became thicker,and formedlaterals ment (Torrey, 1976). The addition to the BD and root buds (Fig. 4). Differentconcentrations mediumof 10 ppm GA3, whichwas essentialfor of myoinositolwere tested as a supplement seed germination(Del Trediciand Torrey,1976), to the BD mediumfor theireffectiveness on root had a slightstimulatory effect (Fig. 1). Indole- elongationin Comptonia. Both 0.1 and 1.0 ppm acetic acid (IAA) at 0.1 and 1.0 ppm inhibited myoinositolelicited a suboptimalresponse. Ten main axis elongation,but stimulatedlateral root ppm and 100 ppm myoinositolproduced essen- initiation.Zeatin at 0.1 and 1.0 ppm also had an tially the same rate of elongation. Higher con- inhibitoryeffect on root elongation. centrationsof myoinositolwere probably supra- A dramaticimprovement in rootelongation was optimal. Combinationsof myoinositoland other shownupon the additionof 100 ppm myoinositol growthsubstances (GA3, IAA, and zeatin) were to the medium (Fig. 2), producingan average added to the culturemedium (Table 1A). Al- elongationof 20 mm/wk,a rate similarto thatof thoughGA3 + myoinositoland zeatin+ myoino- roots of pea (Pisum sativumL.) at 15 mm/wk sitolcaused a higherrate of elongationthan either (Bonner and Devirian,1939), but not as rapid as GA3 or zeatin alone, neithercombination was as 478 AMERICAN JOURNAL OF BOTANY [Vol. 64

TABLE 1. Mean length in cm ? standard deviations (S.D.) of the main axis of cultured roots of Comp- tonia peregrina measured after 6 wk. Each experi- I0 ment included at least 10 initial root tips. A. The in- fluence of combinations of growth substances. B. E8 - The effectof increased sugar concentrationor other s6 - sugar alcohols. _38 Medium Length in cm (? S.D.)

TABLE A BD alone 4.0 ? 1.4 ? 2 4 6 2 46 2 4 6 2 4 6 Time (weeks) 100 ppm myoinositol + 5.0?3.8 Fig. 3. The rate of elongationof isolated roots of 0.1 ppm IAA Comptonia culturedin BD medium+ 100 ppm myoino- 100 ppm myoinositol sitol throughthree successivesubcultures of six weeks + 5.2 1.5 each. c -BD + 100 ppm myoinositol;E1 BD withno 1.0 ppm GA3 additions. 100 ppm myoinositol + 5.8?4.0 0.1 ppm zeatin process was repeated three times in succession 100 ppm myoinositol (Fig. 3). The subculturedroots declined in root ? length and in diameterand often in vigor, but 0.1 ppm zeatin maintainedthe abilityto formroot buds and ap- + 5.5 ?4.5 peared to be thickened.Most of the root growth 0.1 ppm IAA in subculturedroots was in lateralroot formation 100 ppm myoinositol 9.1 ? 3.6 and in the developmentof elaboratelybranched roots (Fig. 5). Because only main axis elonga- tion-was measuredand recorded,an accuraterep- TABLE B resentationof subculturedroot growthwas not 8% Sucrose 5.1?3.5 easily obtained from these experiments.It was 100 ppm D-sorbitol 5.0 ? 3.3 apparent,however, that indefinitesubculture of with 100 ppm Scyllitol 4.9 ? 3.2 Comptoniaroots in BD medium a myoino- sitol supplementwould not be sustained. 100 ppm D-mannitol 3.5 ? 3.2 Secondary thickening-In the BD cultureme- dium withadded myoinositol,Co!mp-tonia roots effectiveas 100 ppm myoinositolalone. IAA was underwentsecondary thickening, increasing 3-4 consistentlyinhibitory to root elongationat the times the diameter of the original excised tip. concentrationstested. The thickenedarea extended along a large por- Carbon sources and sugar alcohols otherthan tion of the proximalhalf of the root as is seen myoinositolwere also tested (Table 1B). When in Fig. 4. The initial excised tip at the time of the sucrose level of the basic BD medium,i.e., inoculation was approximatelythe diameter of withoutadded growthsubstances, was raised from one of the smallerlaterals in the photograph.In 2 % or 4 % to 8 %, the rate of root elongationin- rapidlyelongating roots secondarythickening was creased. This growthrate was not as rapid as in initiatedbefore 6 wk. Thickeningwas also ob- 4 % BD plus 100 ppm myoinositol.Of the sugar served in a slow-growingroot continuallycul- alcohols tested, scyllitol was structurallymost turedin 4 % sucrosemedium for 6 months.Roots similarto myoinositol.However, 100 ppm scyl- developed fromradicle tip-sof embryo-sshowed litol added to BD mediumwith 4 % sucrose had thickeningeven if they were not elongating,but no effecton root growth. Roots culturedin 4 % the thickeningprocess took longer. Actively sucrose BD medium plus 100 ppm mannitolor growingsubcultured roots also thickened (Fig. plus 100 ppm sorbitolhad slow growthrates very 5), althoughthe thickeningappeared more slowly similarto 4 % sucrose BD mediumalone. with each successive transfer.There was often Attemptswere made to establish continuous visible a longitudinalsplitting of the outercortex, cultivationof rootsof Comptoniaby repeatedsub- especiallyon the proximalend of the root. The cultureas has been done in tomato,Lycopersicon thickenedareas frequentlyturned green when the esculentumL. (White, 1938), Convolvulus(Tor- culturedroots were placed in low-intensitywhite rey, 1958) and otherroots. At the end of a cul- fluorescentlight. ture period of 6-8 wk, one-centimetertips were An anatomical study was made of the thick- removed from activelygrowing roots and were ened roots. Figure 6 shows a cross sectionof an subculturedin BD + 100 ppm myoinositol.This unthickenedportion of a root withprimary xylem April, 1977] GOFORTH AND TORREY-CULTURE OF ROOTS IN COMPTONIA 479

Fig. 4, 5. Culturesof isolatedCornptonia roots. 4. A root culturedin 100 ppm myoinositolfrom a radicletip excisedfrom an embryo.The arrowsindicate root buds. X 1.5. 5. Three subculturedroot tipsgrown in BD + 100 ppm myoinositol.The arrowsindicate root buds. X 1.5. arrangedin a tetrarchpattern. The primarycor- Figure 9 shows a cross sectionof a Comptonia tex is intact and contains many intercellular root withthe apical meristemand primordia spaces. A cross sectionof an older regionof the of the root bud clearlyvisible in longitudinalsec- same root is shown in Fig. 7. Secondaryxylem tion. Comptonia root buds appear to originate formedbetween the arms of the primaryxylem in the secondary cortex opposite a protoxylem is evidence of cambial activity. The pericycle pole. Because the secondarycortex is formedby has undergoneone or more divisions,forming the the pericycleor from pericyclicderivatives, the beginningof a secondary cortex. The primary root buds are endogenousin origin. The vascular cortex has begun to slough off. In roots which connectionof the rootbud to the main root forms become more thickened,a solid core of second- at a later stage than that shown in Fig. 9. ary xylem is evident (Fig. 8). Radial rows of parenchymacells formxylem rays in this region. DISCUSSION-The successfulcultivation of iso- A limitedamount of secondaryphloem occurs to lated rootsof woody species would providea use- the outside of the secondaryxylem. The second- ful tool forstudying a varietyof problemsin rela- ary cortex is derived from proliferationof the tion to plant formand function.It is interesting pericycle. The suberized and corky outer layers to considerwhy roots of so few species of woody suggestthe activityof a phellogenand the forma- plants have been established in culture. Rela- tion of a bark-likeouter layer. tivelyfew attemptsmay have been made. Dif- ficultiesin inducingseed germinationmay have Root bud formation-Root buds developed on limitedthe sources of sterileroots for the initial the proximal ends of culturedComptonia roots cultures. Isolated roots of woody species may (Fig. 4). Only four other species of cultured have had requirementsfor special factorsessen- roots,Robinia pseudoacacia L. (Seeliger, 1956), tial for growthnot discovered from studies of Convolvulusarvensis L. (Torrey, 1958), Linaria herbaceous roots. The requirementfor myoino- vulgaris(Charlton, 1965), and Isatis tinctoriaL. sitol by isolated Comptonia roots is an example (Danckwardt-Lilliestr6m,1957) have been re- of such a factor. ported to formendogenous root buds regularly. Myoinositolappears to be an essential com- The root buds firstappeared as small primor- ponentfor the growthof isolated roots of Comp- dia along the proximal end of roots culturedin tonia grownin sterileculture. In the firstculture the dark. When placed in light,the shoot elon- period the presence of myoinositoldoubles the gated and formedleaf-like structures which even- growthof the roots and thereaftermakes possible tually expanded into small which turned the subculturingof roots over several passages. green,then dark burgundyin color. The effectiveconcentrations, i.e., 10-100 ppm, 480 AMERICAN JOURNAL OF BOTANY [Vol. 64

w p a i s l s () S i itats n x 2 xylem(sx) A iseviden.Dis sti o a o hee np g f thepericyet r forin

s(cosscino a thickee areaf ct t h e hdwithd i ai s lt. a i t ..':200. 8.. St~~~~Is I......

(sx)habeenfrmedwthraysof.parnchymaellsi thisregion Somesecondaryphloem(sp)isevi

peripheryofte thscreio The seconarycortex (s)a s folrmed.inThc brightlysbirefringentcellshingof th secodar cor- tex containcalcium oxalate crystals.X 100. 9. A cross sectionof part of a root witha longitudinalsection of a rootbud in the secondarycortex. The apical meristem(am) and leaf primordium(lp are well formed. X 100. April, 1977] GOFORTH AND TORREY-CULTURE OF ROOTS IN COMPTONIA 481 indicatethat it servesa role intermediatebetween thickenafter root decapitation. In the absence thatof a substrateand a co-factoror vitamin. In of an apical meristemthe lateral root primordia this respect, myoinositolbehaves in a manner were foundto play an active role in determining similarto that reportedfor the growthin vitro the differentiationof the vascular tissues. It was of some plant callus tissues (Braun, 1958; Mura- assumed that naturallyproduced auxin was in- shige and Skoog, 1962; Shantz, Sugii, and Stew- volvedin theprocess. ard, 1967) and forresponses such as thatof sec- Loomis and Torrey (1964, 1967a, b) found ondarythickening in excisedroots of radishgrown theycould induceisolated radish roots (Raphanus with basal feeding (Loomis and Torrey, 1964; sativus L.) to undergo secondarythickening in Torrey and Loomis, 1967a, b). Recently,Kaul cultureby addingkinetin (5 x 10-6 M) and in- and Sabharwal (1975) reportedmyoinositol to doleacetic acid (10-5 M) to the sucrose and salts be essential for the survival of callus tissue of in the mediumprovided to the root base in a vial. Haworthiagrown in sterilenutrient culture. The furtheraddition of myoinositolat 100 ppm The role of myoinositolin plant metabolismis to the basal feeding medium stimulatedroot not fully understood. According to Loewus growthand greatlypromoted thickening. Myo- (1971), myoinositolserves as an importantsub- inositolalone did not induced thickening.Peter- strate for cell wall synthesis,being oxidized to son (1973) reportedan essentiallysimilar response form glucuronic acid which is polymerizedto in culturedroots of turnip(Brassica rapa L.). polysaccharidesincorporated into the primarycell The initiationof a vascular cambium and the wall. Jung, Tanner, and Wolter (1972) have formationof secondary vascular tissues in cul- presentedevidence which demonstratesan im- turedroots of Comptoniaare unusual in that no portantrole for myoinositolfor the functioning exogenous hormone supply was required. Most of cell membranes. In eitherrole and possibly of the isolated Comptonia roots which were others, a limitingsupply of myoinositolcould started from an embryonicroot tip thickened limit growth. In Comptonia apparentlythe ex- whetheror not theyunderwent active elongation. cised root is unable to synthesizeamounts of Subculturedroots thickenedonly if root elonga- myoinositoladequate to meetits needs forgrowth tion occurred,either in the main axis or in the and must be provided the growthfactor exog- laterals. This thickeningwas slower than that in enously. the embryonicroot tips. This evidence suggests However,from the growthdata presented(Fig. that theremay be some carryover of substances 3), it is clear thatBD mediumsupplemented with fromthe embryowhich cause thickening.How- myoinositolis not a "complete" medium. Con- ever, thickeningin subculturedroots is caused tinuous growthof Comptonia throughrepeated by somethingsynthesized by the root. subculturehas not been possible with this me- Anotherinteresting feature of isolated Comp- dium and a furthersearch must be made forother toniaroots is theirability to formroot buds. Tor- factorsor conditionsto make continuousculture rey (1958) and Bonnettand Torrey (1966) de- of Comptoniaroots possible. scribed in detail the developmentof root buds Growth in roots of Comptonia is difficultto in Convolvulusarvensis L., which differconsid- determinequantitatively because of the complex- erably from those of Comptonia. Convolvulus ity of the branchinghabit (Fig. 4, 5). Measure- root buds are formedon roots possessing only mentsof linear growthof the main axis presented primarytissues and are initiatedby cell divisions in Fig. 1-3 and Table 1 do not representthe in the pericycleopposite a protoxylempole. A growthresponse adequately. Furthermore,with meristematicdome of cells is organized at the repeatedbranching, the lateralroots become pro- outerperiphery of the primordium.The firstxy- gressivelyfiner, easily damaged in handling,and lem elementsare initiatedobliquely to the xylem difficultto use for subculture.An improvedme- of the root. By two weeks the bud apex and its dium may increasethe vigorof subculturedroots leaf primordiahave penetratedthe outer cortex and theirbranches so as to obviatethese problems. and epidermisof theroot. The formationof secondarytissues in cultured The root buds on Comptonia roots, although roots is an interestingoccurrence and provides ,not arisingfrom primary tissue, are also endog- another experimentalsystem for studyingthis enous in origin. They arise opposite a proto- developmental process. Dormer and Street xylem pole in the secondary cortex which is (1948) firstobserved secondary thickening in an derivedfrom the pericycle. At the time of bud isolated tomatoroot grownin continuousculture initiationthe root has enlargedby the formation for 6 months,a reportvery similar to our ob- of secondarytissues and the outerprimary cortex servationof a 6-month-oldComptonia root which has been split and sloughed off. The vascular had thickenedafter culture in 4% sucrose BD connectionof the root bud develops afterthe for- medium. There have been reportsof secondary mation of the apex and leaf primordiaand con- thickeningin culturedroots in response to ma- nects to the secondaryxylem of the root. nipulationor additionof growthregulators. Tor- Root buds play an importantrole in thevegeta- rey (1951) reportedthat cultured pea rootswould tive propagationof many plant species. Peterson 482 AMERICAN JOURNAL OF BOTANY [Vol. 64

(1975) has writtenan extensivereview on the MoMOT,T. S., I. A. ARMAN, S. F. SZMAYLOV,A. M. initiationand developmentof root buds. Root SMIRNOV, AND A. A. YATSENKO-KHEMLEVSKI. 1974. buds allow plants to form large clones and to Amino acid biosynthesisin isolated roots and ter- cover extensiveareas. When the root systemsare minalcallus tissuesof theEuropean fir (Picea abies (L.) Karst.). Izv. Akad. Nauk. SSSR, Ser. Biol. disrupted,the plant has the abilityto formnew 666-671. plants,thus maintaininga strongholdin the area. MURASHIGE,T., AND F. SKOOG. 1962. A revisedme- Vegetativepropagation of Comptoniaby rootbud dium for rapid growthand bioassayswith tobacco formationon horizontallyspreading roots may be tissuecultures. Physiol. Plant. 15: 473-497. the primarymeans of increasein thisspecies. PETERSON,R. L. 1973. Controlof cambial activityin roots of turnip (Brassica rapa). Can. J. Bot. 51: LITERATURE CITED 475-480. BACHELARD,E. P., AND B. B. STOWE. 1963. Growthin . 1975. The initiationand developmentof root vitro of roots of Acer rubrumL. and Eucalyptus buds,p. 125-161. In J. G. Torreyand D. T. Clark- canaldulensisDehn. Physiol. Plant. 16: 20-30. son [ed.], The developmentand functionof roots. BARNES,R. L., AND A. W. NAYLOR. 1959. In vitrocul- AcademicPress Inc., London. ture of rootsand theuse of Pinusserotina roots SEELIGER,T. 1956. tber die Kultur isolierterWur- in metabolicstudies. For. Sci. 5: 158-168. zeln die Robinie (Robinia pseudoacacia L.) Flora BONNER, J., AND P. S. DEVIRIAN. 1939. Growthfactor 144: 47-83. requirementsof fourspecies of isolatedroots. Amer. SHANTZ,E. M., M. SUGII,AND F. C. STEWARD.1967. J.Bot. 20: 661-665. The interactionof cell divisionfactors with myo- . 1942. Culture of isolated roots of Acacia inositoland theireffect on culturedcarrot tissue. melanoxylon. Bull. Torrey Bot. Club 69: 130- Ann.N.Y. Acad. Sci. 144: 335-356. 133. SLANKIS, V. 1947. Influenceof the sugar concentra- BONNETT, H. T. JR., AND J. G. TORREY. 1965. Auxin tion on the growthof isolated pine roots. Nature transportin Convolvulusroots culturedin vitro. 160: 645-646. PlantPhysiol. 40: 813-818. TORREY, J. G. 1951. Cambial formationin isolated - AND . 1966. Comparativeanatomy of pea roots followingdecapitation. Amer. J. Bot. endogenousbud and lateralroot formationin Con- 38: 596-604. volvulusarvensis roots culturedin vitro. Amer. J. . 1956. Chemical factorslimiting lateral root Bot. 53: 496-507. formationin isolatedpea roots. Physiol.Plant. 9: BRAUN,A. C. 1958. A physiologicalbasis for auton- 370-388. omous growthof the crowngall tumor cell. Proc. . 1958. Endogenous bud Nat. Acad. Sci. (U.S.) 44: 344-349. and root formation by isolated roots of Convolvulusgrown in vitro. CHARLTON, W. A. 1965. Bud initiationin excised PlantPhysiol. 33: 258-263. roots of Linaria vulgaris. Nature 207: 781-782. . 1965. DANCKWARDT-LILLIESTR6M, C. 1957. Kinetin-induced Physiologicalbases of organizationand shoot formationfrom isolated roots of Isatis tinc- developmentin the root,p. 1256-1327. In W. Ruh- toria. Physiol.Plant. 10: 794-797. land [ed.],Encyclopedia of plantphysiology. XV/1. DEL TREDICI, P., AND J. G. TORREY. 1976. On the . 1976. Root hormones and plant growth. germinationof seeds of Comptoniaperegrina, the Annu. Rev. Plant Physiol. 27: 435-459. sweet . Bot. Gaz. 137: 262-268. , AND R. S. LooMIs. 1967a. Auxin-cytokinin DORMER, K. J., AND H. E. STREET. 1948. Secondary controlof secondaryvascular tissue formationin thickeningin excised tomato roots. Nature 161: isolated roots of Raphanus. Amer. J. Bot. 54: 483. 1098-1106. JUNG, P., W. TANNER, AND K. WOLTER. 1972. The , AND . 1967b. Ontogeneticstudies of fate of myo-inositol in Fraxinus tissue cultures. vascular cambium formationin excised roots of Phytochemistry11: 1655-1659. Raphanus sativus L. Phytomorphology17: 401- KAUL, K., AND P. S. SABHARWAL. 1975. Morpho- 409. genetic studies of Haworthia: effects of inositol on ULRICH, J. M. 1962. Culturalrequirements for growth growthand differentiation.Amer. J. Bot. 62: 655- of excisedponderosa pine roots. Physiol.Plant. 15: 659. 59-71. LOEWUS, F. 1971. Carbohydrate conversions. Annu. WHITE, P. R. 1938. Cultivationof excised roots of Rev. Plant Physiol. 22: 337-364. dicotyledonousplants. Amer. J. Bot. 25: 348- LooMIs, R. S., AND J. G. TORREY. 1964. Chemical 356. control of vascular cambium initiation in isolated ZIEGLER, H., AND R. HUSER. 1963. Fixationof atmo- radish roots. Proc. Nat. Acad. Sci. (U.S.) 52: spheric nitrogen by root nodules of Comptonia 3-11. peregrina.Nature 199: 508.