Growth and Organized Development of Cultured Cells. II.

Organization in Cultures Grown

from Freely Suspended Cells
Author(s): F. C. Steward, Marion O. Mapes and Kathryn Mears
Source: American Journal of Botany, Vol. 45, No. 10 (Dec., 1958), pp. 705-708
Published by: Botanical Society of America
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 GROWTHAND ORGANIZEDDEVELOPMENTOF CULTURED CELLS.
GrownfromFreelySuspendedCells'
inCultures
IL Organization
F. C. STEWARD, MARION 0. MAPES, AND KATHRYN MEARS

THE FIRST of thisgroupofpapers (Stewardet al., developsa ring,or hollowsphericalsheath,of cam-

1958) describedthe various ways in whichfreely bium-likeelements(c.f. Gautheret,1956, fig. 8)
suspendedcells fromcertaindicotyledonous plants whichenclosesa mass of cells; thesecellsnow lave
growand multiply to forma relatively
unorganized onlylimitedaccessto theexternalmedium.This can
nmulticellular
mnass.The growthin questionoccurs be seen in fig.3, 4.
under prescribedand controllednutritionaland It appearsthatso longas all thecells of the cul-
environmental The purposeofthispaper
conditions. turehave freeaccess to the externalmedium,they
is to showhoAvthegrowthmaybe carriedforward growin a randomand independent fashionby one
intotheformation of rootsand shootsand, in fact, or otherof themethodsalreadydescribed(Steward
thedevelopment of wholeplants. et al., 1958). The ringof cambium-like tissuede-
DIFFERENTIATION TO FORM ROOTS.-Tissue cul- velops,however,as if in responseto an injuryor a
turesgrowndirectlyfromn cambium-free explantsof wound,underthe stimuliderivedfromthe forma-
differentiated carrotrootphloemin thebasal medi- tion of these lignifiedelements.The cells in this
umsupplemented withcoconutmilkdo notnormally cambium-likeregion are separated from the ex-
formroots. On the veryrare occasionswhenthis ternal medium,and they develop in a nutrient
has occurred,it mayhave been due to thepresence conditionwhichis controlledby the closelypacked
of a preformed rootinitialin the explant. At any zone of cells and thetissuewhichtheyenclose. In
rate,root formnation occursextremely infrequently fact,thedividingcellslie alonga gradientfromthe
whenthe cultureis startedfromfreshlyexplanted dead cells within,to the nutrientwithout,which
phloemtissue. On theotherhand,theminutetissue containsthecell divisionstimuli.In thiscambium-
culturesformedby the development of cell aggre- or pericycle-like region,a root apex formsand it
gates fromthese freelysuspendedand frequently subsequently growsoutthroughthetissuemassinto
sub-cultured cellswilldeveloprootswithgreatease. the surroundingmedium (fig. 5)- These events are
Thismaybe due to thedisappearanceofsomefactor not demonstrably differentfromthe normalorigin
antagonisticto the formation of an organizedroot of lateralroots. Whensuchrootinitialsform,they
apical mieristem thatis presentin the centralcore growapace and the morecallus-likegrowthof the
of tissue in the explantremovedfromthe carrot original colony of cells tends to be suppressed.
root. Or, it may be said that the differentiatedWhen grown in this way, on culturesrevolved
phloemtissuemustfirst"de-differentiate"-whatever around a horizontalaxis, the roots emergein all
thatmaymean. Whateverexplainstheease of root directions(fig. 6). Longitudinalsectionsof roots
formationin these small aggregates,which are so formedshow thattheyare normaland develop
grownfromculturedcells,the eventsthat lead to apparently normalprotoxylem tissue.
rootinitialscan be illustrated in fig.1-5. FORMATION OF SHOOTS.-Whencultureflasksor
A rootinitialdoes not formuntilthe cell aggre- tubes are inoculatedby liquid suspensionswhich
gate or colonyreachessuch a size thatthe inner containfreelysuspendedcells or small cell aggre-
cells of themass behavedifferently fromthe outer tates,theyformverylargenumbersof freelygrow-
cells. A sign of thisis the presenceof a new type ing colonies,and manyroot initialsmay originate
cell in the centralregionof the culture(fig. 1) ; in the mannerdescribed.The conditionsmostcon-
thiscellloses itscontents, beconming somewhatligni- duciveto rootformation have notbeen investigated
fiedand forming a tracheid-like elementof thekind fully,thoughsome earlyevidenceseemedto indi-
so oftenobservedin planttissuecultures(Gautheret, cate thatin a culturewhichis proneto formroots
1956). A single xylemelementis frequently fol- this occurredmore readilyin the dark and more
lowed by similarones in close association. It is readilyif the calciumcontentof the mediumwas
interesting that theyformin a tissue whichwas reduced.Furthermore, it has beennotedthatcertain
wholly derived originally from the secondary active growth-promoting fractions,isolated from
phloem. Surroundingthese nests of lignifiedele- extractsof immaturecorn (Shantz and Steward,
ments,whichlose theircontents(fig. 24), there 1957) also tendedto fostercopiousrootformation.
However,oncea rootinitialhas formed, it continues
1 Received forpublicationJune 1, 1958. to grow,and thereafter
This workformspart of a programiof researchwhich has
the growthof the tissue
been supported by grants to one of us (F.C.S.) fromthe cultureis retarded,or suppressed. If, however,
National Cancer Institute,National Institutes of Health, cultured cell aggregateswithrootsalreadydeveloped
United States Departmentof Health,Education,and Welfare. are transplanted to nutrient
agar in flasks,complete
[The JournalforNovember (45:653-704) was issued November22, 1958]
AMERICAN JOURNAL OF BOTANY, Vol. 45, No. 10, December 1958

705

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706 AMERICAN JOURNAL OF BOTANY [Vol. 45

"|~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
..............
..
444 ~~ ~ ~ ~~4

...
,'~4 8 .. 9 ti

I. RI

Fig. 1-15. Carrot. Fig. 1-4. Stages in the formationof "nodules," or growthcenters,which enclose "nests" of lignified
elements.Fig. 1 shows the firstlignifiedelementand adjacent cell division; fig 3 shows complete ring of dividing tissue

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 December,1958] STEWARD ET AL.-DEVELOPMENT OF CULTURED CELLS II. 707

withbasal mediumand coconutmilk,the cultures because cells withdrawnfromthe phloemof the

can be indefinitely continued.Underthesecircum- storagecarrotrootand whichhave passed through
stancesa copious growthof rootsoccurs. Indeed, manytransfers in whichtheywerereducedto the
if culturesremainin the normalrotatingculture singlecellularstate,have developedintocell aggre-
tubesin whichexplantsare commonlygrown,the gates,whichhave, in turn,differentiated to form
roots may grow until theyvirtuallyfill the tube rootsand, whentransplanted, have givenrise also
(Steward and Shantz, 1955, fig. ld). If, how- to shoots and to a secondarilythickenedstorage
ever,tissueaggregateswhichhavebeenrearedfrom carrotroot.
freecellsare transplanted to agar mediatheymay, A curious,but suggestive, observationwas made
as theygrow,also formbuds and shootsspontane- on the way the secondarilythickenedcarrotroot
ously. grewin culture(fig. 13-15). The surfaceof the
The shootswhichfirstemergehave leafyappend- enlargingorganwhichwas in contactwiththeagar
ages which,like primitive leaves,tendto be entire mediumcontainingcoconutmilk grewirregularly
(fig.7), thoughlatermoretypical,muchdissected, at itsbase,withgreencallous-like swellings(fig.13).
carrot leaves develop (fig. 11, 12)- Again, the The side of thestoragerootwhichwas away from
stimuliwhichpromotethedevelopment of buds and the medium,and, therefore, not in directcontact
shootshave not yet been fullyinvestigated.From withcoconutmilk,grewnormally, forming a broad
the frequency withwhichthisdevelopment occurs, band of secondaryphloem,rich in carotene,as
it would appear thatthe irregular,randomlypro- seen in transversesection (fig. 14). This tissue
liferatedculturesthatdevelopfromthe freelysus- appearednormalin everyway, in contrastto the
pendedcellsdo thisspontaneously and withcompar- liquid-cultured,unorganizedtissue which never
ative ease whentheyare furnishedwiththe basal achievesthis rich,orange-redcolor or contentof
mediumplus coconutmilk on a stationary, semi- carotene. The only other abnormalitywas some
solidmedium.No doubttheshootis moredependent greencolor in the xylem,whichwas seen in the
on regularorientation to gravitythan is the root. longitudinal section(fig.15); thisis a notuncom-
The looselyproliferated culture,whichoriginated monfeaturenearthecrownofnormalcarrotplants,
fromaggregatesgrownfromfree cells and later especiallywhentheyhave been grownin the light.
transfered to agar, presentsthegeneralappearance The obvioussuggestionis thatdirectcontactwith
shownin fig.7. From such a mass manyminute the stimulito growth,which are in the coconut
plantletscan be dissected,such as thoseillustrated milk,leads to cell proliferation (fig.13) but,when
in fig.8-10. thesestimuliare modifiedby theintervening tissue
REGENERATED PLANTLETS: SIMILARITIES TO YOUNG of the growingroot,apparently normalsecondary
EMBRYOS.-In the regeneratedplantlets,the axis, growthmay occur (fig.14, 15).
with shoot and root apices, is completelyestab- THE STIMULI TO DEVELOPMENT.-Much remains
lished (fig.8). The impressionis inescapablethat to be done to definethe variablesand the stimuli
each of the many nodule-likegrowing centers whichreg.ulateeach of the definitive steps in the
(fir. 5) whichdevelopin the culturedmass can organizationof cellular aggregatesto formroots
firstformroots; then,if transferred to a stationary and shoots. It is suggestivethatthe effective nutri-
agar medium,they will formshoots. Thus, the entconditionsforthisdevelopment also furnishall
sphericalmassesof culturedcells,enclosedby their the nutrientsand growthfactorsthat normally
sheathof cambium-like initials (fig. 3, 4), really nourishimmatureembryos.Thus, it is now clear
behavelike a proembryo;theyformbothrootsand that parenchymacells, whichare already far ad-
shootsand, significantly enough,do this in a fluid vanced towardmaturityin the plant body, may
(of anotherspecies) that normallynourishesim- returnin culture,underappropriatenutrientcon-
matureembryos.Figures7-12 showvariousstages ditions,to the dividingstateand, as theydo this,
in the organizationof the shootin culturesof this theycan eventually recoverthetotipotency thatwas
sort. Even more surprisingly, it may be observed originallyinherentin the egg. However,no single
thata secondarily thickened storagecarrotrootmay parenchymacell can directlyrecapitulatethe fa-
grow in the tissue mass whichhad originatedin miliarfactsof embryology, but,throughtheforma-
the mannerdescribed,frommaterialthatbad been tionfirstof an unorganizedtissueculture,whichis
frequently sub-cultured and reducedto thefreecell in fact a colony of dividingcells, the necessary
state(fig.12, 13). degreeof organizationis recaptured,firstto form
Thus the cycleof development is now complete, roots and thento formshoots.

enclosing"nests" of lignifiedelements; fig.4 shows the beginning of organizationin a nodule leading to root formation.-
Fig. 5, 6. Emergenceof roots fromtissue cultures.-Fig. 7, 11, 12. Stages in the growthof carrot plants on agar.-Figs.
8, 9, 10. Stages in the developmentof young plantlets,which originatefromindividual nodules (fig. 8 shows veryyoung
stage in this development).--Fig. 13. Carrot plant with storage organ.-Fig. 14. Transversesection of cultured storage
root shows callus-likegrowthin contactwith the mediumplus coconut milk and normal secondaryphloem on side away
fromthe medium.-Fig. 15. Longitudinal section of cultured storage root.

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708 AMERICAN JOURNAL OF BOTANY [Vol. 45

Oile maywellask whythecellsof tissueexplants, originatesby the growthof freelysuspendedcells

withdrawn fromthe phloemof a carrotroot,will thathave been obtainedin the mannerpreviously
not as readilyorganizeto formrootsand shoots, described(Stewardet al., 1958). For carrottissue,
eventhoughtheycan be broughtby coconutmilk it is demonstrated thatnormalrootsmay arise in
into a rapid state of cell divisionand may form theliquid mediumand, once thesehave originated,
verylarge callus-likemasses if theyare grownon thecultureswilldevelopshootswhentheyare placed
an appropriatemedium. The full explanationof on a semisolidagar mediumand are enabled to
the contrastedbehavior of the cells which have growin a polarizedway in stationaryculturesex-
passed throughthe freely suspended condition posedto air. The stagesby whichplantletsdevelop
and thecells of theexplantsfromwhichtheywere are illustrated, and eventually secondarythickening
originallyderived requires furtherinvestigation. occursarounda newlyformednestof xylemcells,
Thoughthe processmay be describedas "dediffer- afterwhichtheremayoccurproductionof a carrot
entiation,"this termis hardlyilluminating.It is, storage root. Thus the cycle from carrot root-
however,interesting to recallthatin theearlywork phloem to free cell, to carrot root, to carrot plant
ofVan Overbeeketal. (1941), someyoungembryos has been completed. The essential stages in this
wereinducedto growin an unorganizedprolifera- reorganization involve: (1) The formation,within
tive fashionby the use of coconutmilk. In the the culturedmass, of a spherical sheath of cambium-
presentinvestigation,already differentiated cells like cells which completelyencloses a nest of ligni-
have been inducedto growagain and eventually to fied elements so typically observed in tissue cul-
produce structuresthat normallyoriginatefrom tures; (2) this spherical nodule, or growing center,
the embryo. This surelymeans that the coconut plays a part equivalent to the pro-embryoin normal
milkcontainstheinherent stimuliand nutrients that development,for it can give rise eventuallyto roots
make for activegrowthby cell division,but that and subsequentlyto shoots; and (3) the formation
the progresstowardorganizedgrowthrequiresthe of a shoot initial occurs at a point diarm-etrically
growingsystemto acquire a measureof independ- opposite to the emergence of the root apex, pro-
ence fromthe coconutmilk stimuliwhich,if un- ducing a simple, embryo-likestructurereminiscent
regulated,lead to unorganizedgrowth.This inde- of that which occurs in normal development. The
pendencefirstoccursthroughthe formationof an stages in the developmentof a plantlet into a com-
unbrokensurfaceof dividingcambium-like cells, pletely organized plant with storage root are illus-
surrounding a regionin whichsomecellsof a tissue trated. It is emphasized that this orderly develop.
culturematureand die. Withinthis "walled-off" ment becomes possible when cells are enclosed
zone,somecellsare confined and a rootinitialforms wvithin,and limited by the restraints probably
(fig. 7) and, thereafter,having formed roots, more physiological than physical-of, this wall of
the culturemay grow in responseto the coconut cambium-like cells which effectivelycuts off the
milkand also formbuds. It is difficult, therefore, internalcells fromdirect access to the coconut milk
to avoid the conceptthat the root initial must stimulithat cause random proliferation. Wherever
originate,underrestraint, in a controlledenviron- the coconut milk has direct access, more random
ment,withina ring of cells which permitonly callus-likegrowthoccurs.
controlled access to thecoconutmilkstimuli;buds,
in turn,formonlyafterthisspecial noduleof cul- DEPARTMENT OF BOTANY

turedcells has respondedto the culturemediumin CORNELL UNIVERSITY

a polarizedmannerbroughtabout by the presence ITHACA, NEW YORK

of roots.
LITERATURE CITED
It will be a major landmarkwhenthe fertilized
eggis removedfromtheenvironment oftheembryo GAUTHERET, R. J. 1956. Histogenesis in plant tissue cul-
sac and the facts of early embryogenycan be tures. Jour. Nat. Cancer Inst. 19: 555-573.
recapitulated withtheproductionof leaf,stem,and SHANTZ, E. M., AND F. C. STEWARD. 1957. The growth-
rootundercultureconditionsin a synthetic medium. stimulatingsubstances in extracts of immature corn
a progress report. Proc. Amer. Soc. Plant
The presentseries of papers show thatfree-living, grain: Physiol.,p. 8. A. I. B. S. meeting,Stanford,California.
disassociatedcells maybe caused to growin media STEWARD, F. C., MARION 0. MAPES, AND JOAN SMITH. 1958.
containing coconutmilkso thattheyachievesimilar Growth and organized developmentof cultured cells.
ends to thosewhichresultfromthe growthof the I. Growthand divisionof freelysuspended cells. Amer.
zygote,fortheyproducerootsand shootsand even Jour.Bot. 45: 693-703.
secondarilythickened storageorgans. However,the - AND E. M. SHANTZ. 1955. The chemical induction
freelysuspendedcells do not do this,as it were, of growthin plant tissue cultures. I. Methods of tissue
directly,but onlymoredeviouslythroughthe sum- culture and the analysis of growth. In The chemistry
and mode of action of plant growthsubstances. WAIN,
mationoftheeventswhichhaveherebeendescribed. R. L., AND F. WIGHTMAN (ed.). Butterworths.London.
VAN OVERBEEK, J., M. E. CONKLIN, AND S. F. BLAKESLEE.
SUMMARY
1941. Factors in coconutmilk essential for growthand
This paper describestheway in whichorganiza- developmentof very young Datura embryos. Science
tionmay developin a culturedmass of cellswhich 94: 350, 351.

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