Growth and Controlled Morphogenesis in Pea Root Callus Tissue Grown in Liquid Media Author(S): John G

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Growth and Controlled Morphogenesis in Pea Root Callus Tissue Grown in Liquid Media Author(s): John G. Torrey and Yoshio Shigemura Source: American Journal of Botany, Vol. 44, No. 4 (Apr., 1957), pp. 334-344 Published by: Botanical Society of America Stable URL: http://www.jstor.org/stable/2438385 . Accessed: 23/08/2011 15:27

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http://www.jstor.org 334 AMERICAN JOURNAL OF BOTANY [Vol. 44

MCGONAGLE, MOIRA P. 1944. Culturesof excisedlegumi- plantin determiningthe infection of theroot by the nous roots. Nature153: 528-529. nodule-formingbacteria. Proc. Roy. Soc. LondonB. NEAL, 0. R., AND R. H. WALKER. 1935. Physiological 104: 481-492. studieson Rhizobium.IV. Utilizationof carbona- . 1936. The actionof sodiumnitrate upon the in- ceous materials.Jour. Bact. 30: 173-187. fectionof lucerne root-hairsby nodule bacteria. RAGGIO, M., AND NORA RAGGIO. 1956a. Relacionentre co- Proc. Roy. Soc. LondonB. 119: 474-492. tiledonesy nodulaciony factoresque la afectan. WHITE, P. R. 1943. A handbookof planttissue culture. Phyton(Argentina) 7(2): 103-119. The RonaldPress Co. New York. , AND . 1956b. A new methodfor the culti- WILSON, J. K. 1917. Physiologicalstudies of Bacillus vationof isolatedroots. Physiol.Plantarum 9: 466- radicicolaof soybean(Soia max Piper) and of fac- 469. tors influencingnodule production.Cornell Univ. SEPPILLI, A., M. SCHREIBER, AND G. HIRSCH. 1941. L'infezi- Agric.Expt. Sta. Ithaca Bull. 386. one sperimentale"in vitro"delle colturedi radici . 1931. Nodule productionon etiolatedvetch seed- isolatedi Leguminose.Ann. Acad. Brasil.Scien. 13: lings.Phytopathology 21: 1083-1085. 69-84. WILSON, P. W. 1940. The biochemistryof symbioticnitro- THORNTON, H. G. 1929. The role of the younglucerne gen fixation.Univ. Wisconsin Press. Madison.

GROWTHAND CONTROLLEDMORPHOGENESIS IN PEA ROOT CALLUS TISSUE GROWNIN LIQUID MEDIA JohnG. Torreyand YoshioShigemura

IN RECENT YEARS considerableinterest has cen- using tissueculture methods, careful identification tered in the nutritionalrequirements of isolated of both morphologicaland anatomicalorigin of fragmentsof higherplant tissues cultured in vitro. the tissuemay be a matterof considerableimpor- From the early work of White (1943) and of tance. Thus, callus tissuederived from roots may Gautheret(1942), it has been knownthat tissues be expectedto differfundamentally in inherent derivedfrom the immediatemorphological region morphogeneticcapacities fromstem callus tissue. of the vascularcambium of eitherstem or rootare These differencesmay be perpetuatedin culture particularlysusceptible to culture in a suitable or they may be modifiedby manipulationof the nutrientmedium. Much informationhas been mediumor by the passage of time. gained concerningthe specificnutritional require- Duringthe past severalyears, studies have been mentsof a numberof callus tissuesderived from made.ofthe particularnutritional conditions neces- the vascular cambiumregion of a wide varietyof sary for continuedmeristematic activity in the plants (Gautheret,1955b). rootmeristem of isolatedpea rootsgrown in vitro Most of the callus tissueswhich have been cul- (Torrey,1954) and for the initiationof cell divi- tivatedin vitrohave been derivedfrom secondary sions leading to lateral root formationin isolated tissues of stems. Relativelyfew callus tissues of pea rootsegments (Torrey, 1956). Because of our root originhave been studied. One notableexcep- interestin cell divisionand its biochemicalcon- tion is carrottissue whichhas been grownexten- trol in pea roots grown in culture,it appeared sivelyin culture,but even here precisedistinction desirableto us to isolate tissuesfrom the vascular betweenroot and stem (hypocotyl)tissue is diffi- cambiumregion of pea roots and to study the cult to make and frequentlyhas not been made. nutritionalconditions necessary for continuedcel- Only in a fewcases have rootcallus tissuecultures lular divisionsin such tissues. Once estab-lished of distinctlyroot origin been described. Skoog in culture as a root callus tissue, it was hoped (1944) reportedthe spontaneousformation of root thatthe peculiarconditions giving rise to the initi- callus by culturedroots fromhybrid tobacco tis- ation and continuedactivity of the secondary sues in culture. Jagendorfet al. (1952) describe meristemin vivo mightbe discoveredand that a a callus tissueproduced from cabbage roots,Kand- studyof the inherentmorphogenetic capacities of ler (1950) fromsunflower roots in culture,Tryon the rootcallus tissueitself might be made. In the (1955) fromtobacco roots,Nickell (1955) from experimentalwork to be reportedhere, an account rootsof sweetclover, and Black (1947) and Nickell is givenof theestablishment on a complexnutrient (1954) the abnormalvirus tumor tissue produced mediumof a callus culture.from tissues isolated by sorrelroots. fromthe vascular cambiumregion of pea roots. In studyingproblems of plant morphogenesis During the course of this study,the oallus tissues have been grownin bothsolidified agar media and 1Received for publication October 16, 1956. in liquid media withconstant agitation. Under the This investigationwas supportedin part by research latterconditions two morphologicallydistinct tis- grantnumber RG-2861 to the seniorauthor from the Na- tionalInstitutes of Health,U. S. Public HealthService. sues have becomeevident whose occurrenceis de- The authorsare indebtedto Mr. VictorDuran for his pendent upon the constituentsof the nutrient photographicwork. medium. April,1957] TORREY AND SHIGEMURA-GROWTH IN CALLUS 335

Establishmentof the pea root callus tissue in but turnedbrown and died if thepieces were main- culture.-Five-mm.root tips excised from 48-hr. tained on the auxin-containingmedium used in germinatedpea seeds,variety 'Alaska,' were trans- the initial treatmentwhich had given rise to the ferred aseptically to Petri dishes containinga cambium. This medium,free of auxin, is known modifiedBonner medium (Torrey, 1954) with0.5 to support the continuedactivity of the apical per centagar and 10-5M indoleaceticacid (IAA) meristemof pea roots throughat least a number at pH 5.0. In certaincases, the radicle of the pea of weeklytransfers during which growth in length seed was excisedaseptically from the dryunsoaked averages between40 and 60 mm. The medium seed and transferredto the IAA medium.The ex- containsas growthfactors essential to rootelonga- cised root tips were maintainedon the auxin tion the vitamins,thiamin and nicotinicacid as mediumin the dark at 25C. for 5-7 days. well as the essentialmicronutrient elements. For In the auxin medium,elongation of the roottips continuedmeristematic activity in the vascular was markedlyinhibited. By the fifthday the basal cambium region,however, additional factors.ap- regionof the excisedroots had increasedin diame- parentlyoriginally present in the root tip excised ter to almost twice the original dimensionand fromthe germinatingseed, mustnow be provided lateral roots were evidentin this enlarg.edregion fromthe externalnutrient medium. (Torrey,1950, 1951). Histologicalpreparation of -Experimentswere made in which the tiss4e such rootsshowed that the increasedroot diameter pieces containingthe vascular cambium region was largely attributableto extensivesecondary were testedon a large numberof complexmedia. activityfrom a well-establishedVascular cambium. The medium most effectivein producingrapid By the seventhday, the vascular cambium,which proliferationof cells which originatedin the im- had originatedin typical fashion by periclinal mediatearea of the vascular cambiumzone, was divisionof cells in the areas betweenthe primary a modifiedBonner agar mediumwith 4 per cent phloembundles and the primaryxylem, was con- sucrose,to whichhad been added, prior to auto- tinuousand had formeda triangularcentral bun- claving,1 g./l. Bacto yeast extract (Difco) and dle of xylem (fig. 1). 10-5M 2,4-dichlorophenoxyaceticacid (2,4-D) . Using a sharp scalpel of the replaceableblade Tissue pieces which showed proliferationmain- type,the basal end of the rootwas cut into 1-mm.- tained a golden yellow colorationand could be thickcross-sections which were placed on the agar seen under the dissectingmicroscope to manifest medium. The corticaltissues were cut away from a "boilingup" of cells in thecambial zone. Xylem the central cylinder tissues under a dissecting tissueof such pieces tendedto turn brownin all microscopeand triangularwedges were cut along media and did not contributeto the growthof the the radial lines of the primaryxylem poles. These callus culture. Pieces showingproliferation from wedges included tissues of the primaryand sec- the vascular cambiumregion were transferredbi- ondary xylem,vascular cambium,secondary and weeklyto the same mediumfreshly prepared until primary phloem, pericycle, and endodermisas a large roundedmass of proliferatingtissue had well as damagedcortical cells. Each wedge-shaped been formed(fig. 2). Such callus masseswere cut piece was transferredaseptically to a Petri dish into smallerpieces using a sharp scalpel, and the of modifiedBonner medium containing 0.5 per cent small pieces were subcultureduntil a clone of cal- agar and various additionsof growthfactors and lus tissue sufficientfor experimentalpurposes was metabolites,and the pieces were culturedin the established. dark at 25?C. Of the auxins testedfor effectivenessin estab- An alternateprocedure which was foundequally lishingthe pea root callus tissuein culture,2,4-D successful,although much less convenient,was to was foundto be the mosteffective. a-naphthalene secure vascular cambial tissues from sectionsof acetic acid in the presenceof 1 g./l. yeastextract, mature regions of untreatedweek-old pea root also stimulatedcallus development.IAA in the seedlings (see Popham, 1955). In such seealing presenceof yeastextract, was inactive. roots,manipulation is moredifficult. Othercomplex growth factor mixtures were also It is a matterof considerableinterest that only testedin the presenceof 10-5M 2,4-D. Yeast ex- initial root tips taken fromthe germinatingseed tractwas consistentlymost effective in establishing respondto auxin-treatmentby producinga vascu- the callus. Peptone at 1 g./l. when added to the lar cambium with extensivesecondary activity. modifiedBonner medium in the presenceof 10-5M Five-mm.-tipsexcised as firsttransfer tips, i.e., 2,4-D permitteda slow developmentof root callus frominitial root tips which had been grownon tissue from wedge-shapedpieces. Similar slow auxin-freemedium for one week to an average developmentwas achieved in the presenceof 15 lengthof about 60 mm.,when placed on a modi- per centautoclaved coconut milk and 10-5M 2,4-D. fied Bonnermedium containing 10-5M IAA, show Because of the consistentlybetter response of the no secondaryactivity and no vascular cambium. tissuesto the mediumwith yeast extract and 2,4-D, The chemicalnature of the startingmedium.- it was decidedto use thismedium as the basis on The wedge-shapedroot pieces containingthe vas- whichto worktoward a syntheticmedium. Clonal cular cambiumdid not continuecellular divisions materialwas maintainedon agar mediumcontain- 336 AMERICAN JOURNAL OF BOTANY [Vol. 44

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Fig. 1-7.-Fig. 1. Transverse section of isolated pea root grown for 7 days on modifiedBonner medium containing 10-5~M IAA, show'iugextensive secondary tissues produced by a vascular cambium. X 140.;-Fig. 2. Pea root callus April,1957] TORREY AND SHIGEMURA-GROWTH IN CALLUS 337 ing 1 g./l. yeast extractand 10-6M 2,4-D with bi-weeklytransfer and subcultureto freshmedium. The callus tissuesgrown on agar media were ob- servedto be of two types: eithersmooth, rounded .j 600 solid masses of tissue (fig. 2) or loose, friable masses which readily broke apart when handled (fig. 3). Usually, spherical masses of approxi- ~~~/ \ ~~~~~~~10-5M 2,4-a mately5 mm. diameterwere used as subcultures. 400 Establishmentof clonal materialin liquid culture.-For quantitativestudies it was desirableto culturethe root callus tissue in a liquid medium. Preliminaryexperiments showed that the callus -J 200 tissue would continueproliferation in submerged S :c 20 _ _ __ *O. \-\ 2,4-204-\ culturein a 50-ml.volume of yeast extract-2,4-D mediumper 125-ml.Erlenmeyer flask if the flasks were constantlyagitated. Routineculture of the 0 0.01 0.1 1.0 10.0 callus in liquid mediumwas achievedby use of a < horizontal-typeshaker which producesa vigorous CONCENTRATIONOF YEAST EXTRACT(GM/L) swirlingmotion of the contentsof the flasks.Indi- Fig. 8. The averageincrease in wet weightin mg. of vidual experimentsof 200 or moreflasks could be pea rootcallus tissuegrown for 8 weeksin liquid medium run readilyon the shaker. Flasks weremaintained containingdifferent concentrations of yeast extractwith at 25?C. in the dark withoccasional brief periods and without2,4-D. of whitefluorescent light for observations. Clone 33 whichwas used throughoutthe experi- were determinedafter drying at 950C. to constant ments reportedhere was derived from a single weight. wedge-shapedpiece of pea roottissue excised from The interactionof yeastextract and 2,4-D in the the cambial regionof anwIAA-treated root at the growthof the callus tissise.-It was apparentquite 5thday. The calluswas establishedon agar medium early that the yeast extract was essential for the containing1 g./l. yeast extractand 10-5M 2,4-D, maintainedgrowth of the callus in cultureand that was transferredweekly to freshagar mediumfor 2,4-D had a markedsynergistic effect in the pres- 4 weeks and was then culturedin liquid medium ence of yeast extract. Experimentswere made to containing1 g./l. yeast extractand 105M 2,4-D. determinethe optimumconcentrations of these As the initial inoculumgrew in size, increasingas two componentswhich would produce maximum a solid, sphericalmass of tissue,it was quartered growth.In fig. 8 are presenteddata showingthe or furthersubdivided into 3-4-mm. diameter pieces effectof differentyeast extractconcentrations on at approximately4-week intervals until many hun- the increasein wet weightin the presenceand in dreds of separate pieces were growingin liquid the absenceof 10-5M 2,4-D. The averageincrease culture. These clonal cultures were routinely in wet weight per culture at the end of 8 weeks is maintainedwith 5-8 pieces per 50 ml. of medium presented. per flask. In orderto minimisethe lag in growth The followingfacts are evidentfrom these re- associatedwith the woundingeffects of the separa- sults: (1) In the absence of yeast extract, very tion procedure,the freshlyquartered pieces of little growthoccurs whetheror not 2,4-D is present. inoculumwere usually carriedin the controlme- The growthincreases as the yeast extractconcen- dium (1 g./l. yeastextract and 10-6M 2,4-D) for tration increases up to an optimum of 1 g./l. and 4 weeksat whichtime the spherical5-mm.-diame- then higher levels of yeast extract become toxic. ter pieces were transferred,one per flask,to the From this fact, one can deduce that some com- medium to be tested. Routinely,twenty fl-asks ponent of yeast extract is essential to the optimum were run for each testmedium and half were sac- growth of the callus and is limiting at low con- rificedat 4 weeks,the second half at 8 weeks- centration. (2) In the absence of 2,4-D, yeast for freshand dry weightdeterminations. In cer- extract alone supports some growth of the callus, tain cases, ten flasksamples were takenat 2 week reaching its most effectiveconcentration at 1 g./l. intervals. Fresh weights were determinedafter At 5 g./l. or above, the yeast extract alone is blottingaway excesssurface moisture; dryweights highly toxic. In the presence of 10-5M 2,4.D, type grown on agar medium for 4 months.-Fig. 4-7. Pea root callus tissue cultured in liquid media in flasks. All photographedin 11-cm. Petri dishes.-Fig. 4. Callus culture of the RS type grown in M6 medium for 4 weeks.-Fig. 5. Callus culture started from single piece of RS type callus, grown for 4 weeks in medium containing 10-5M 2,4-D plus 5 g./l. yeast extract. Note large darkened original inoculum with numerous small friable pieces.-Fig. 6. Callus culture of the friable type propagated on medium containing 10-5M 2,4-D plus 5 g./l. yeast extract. Note numerous rounded protuberanceson each piece.-Fig. 7. Pieces of friable callus tissue after transferto modifiedBonner medium lacking both yeast extract and 2,4-D. Note roots which have been formedfrom the small protuberances. 338 AMERICAN JOURNAL OF BOTANY [Vol.44 yeastextract exerts a similarstimulating effect on fromfriable tissues only. Wet and dryweight de- growthwith an optimumat 1 g.il. The auxin terminationsof the friabletissues could be made alone has essentiallyno effecton increase in by determiningthe aggregate weight of the culture weight.The two componentstogether, however, act pieces. However,from such friable pieces it has synergistically.In the presenceof 10-5M 2,4.D, not provedpossible to propagatea uniformclone higher levels of yeast extractare tolerated,al- of the RS type,nor even to returnto the RS con- thoughthe higherconcentrations are less effective dition. Furthermore,it seemsevident that the two in producingincreased wet weight. At 10 g./l. typesof clonal materialrespond differently to dif- yeastextract is consistentlytoxic. ferentnutritional conditions and should in no way Experimentswere then run to establishthe opti- be consideredidentical tissue systems. mum2,4-D concentrationin the presenceof I g./l. Conditionsleading to the friabletype of callus yeastextract. In fig.9 are presentedthe results of growth.-Because of the considerableinterest in wet and dry weightdeterminations of callus cul- the physiologicalbasis of thefriable type of callus tures grown in differentconcentrations of 2,4-D growth,an extensiveseries of experimentswas with a constant yeast extract concentrationof carried out to determinethe nutrientconditions 1 g./l. These resultsare presentedas averagetotal leading to friability.It was soon foundthat the weightper culturewith an initialinoculumT weight conversionof the RS type callus to the friable of 90 mg. wetand 11 mg. dryweight respectively. callus depended upon the relativeconcentrations It is evidentthat maximumgrowth in termsof of yeastextract and 2,4-D in the medium.In com- increasein both wet and dry weightresults from paring the effectsof the differentmedia on fria- a mediumcontaining 10-6M 2,4-D in the presence bilityit mustbe bornein mindthat the nutritional of 1 g./l. yeast extract. At higherconcentrations conditions must be such as to allow some growth of the auxin, lower tissue weightsoccur. In all to occur. In subsequentexperiments, the yeast subsequentexperiments the clonal material was extractconcentration of 10 g./l. was omittedsince maintainedin this medium (M6) and the callus no growthoccurred at any of the 2,4-D concen- grew consistentlyand well as smoothround solid trationstested (e.g., see fig. 8). An intermediate masses of tissue. concentrationof 1.5 g./l. yeast extractwas intro- During the course of these experiments,a very duced. The resultsof these experimentsare sum- disconcertingphenomenon began to show itself. marizedin fig. 10 in termsof averageincrease in The initial inoculum used in each flask was a wet weight (fig. lOa) and average number of spherical piece of callus tissue, quite solid and pieces per culture(fig. lob). woody,of golden color, roundand smoothin ap- Analysisof fig. lOa showsthat the M6 medium pearance. This type of callus (fig. 4) was desig- (1 g./l. yeast extractand 1076M 2,4-D) repre- nated the RS type and could be reproducedre- sents the best medium for wet weight increase. peatedly with large numbersof uniformpieces A second lesser peak occursat 1.5 g./l. yeast ex- when propagatedas describedpreviously in the tractand 10-5M 2,4-D whenboth componentsare M6 medium. at a slightlyhigher concentration than in the M6 In the experimentsdescribed above, however, medium. When used alone, 1.5 g./l. yeast extract in whichvariations of 2,4-D and yeastextract con-i already shows toxicityand no growthoccurs at centrationswere tested,the growthof the callus 5 g./l. yeast extract.These data confirmand ex- tissuewas not always thatof the RS type. Under tend those already presentedwith respectto the certainnutritional conditions, the initial inoculum interactionof 2,4-D and yeast extracton growth showed a delayed proliferation,which occurred in termsof wet weightproduction. sporadicallyfrom localized areas on the surface Referenceto fig.lOb showsthat the peak of wet of the inoculum,so that small nodular pieces of weightincrease is the point of least friabilityin tissuewere formed.These pieces increasedin size these experiments.Thus adoptionof the M6 me- and were frequentlybroken off from the initial diunmfor the maintenanceand propagationof the inoculum.At theend of theexperiment, such flasks RS typeclone was a happy choice. Furtherstudy containedmany small pieces, each growingsepa. of fig.lOb showsthat friable growth occurs under rate fromthe originaltissue mass (fig. 5). Thus two nutrientconditions: i.e., at 1 g./l. yeast ex- the tissue had become friable and grew thence. tract in the absence of 2,4-D and, at the other forthin a fundamentallydifferent manner. Such extreme,at 5 g./l. yeastextract and 10-5M 2,4-D. callus tissueswere designatedthe Friable type. In Neitherof these conditionsis particularlyfavor- fig.4 and 5 the two cultureswere started from the able to growthin weight. In general,one can say samneRS type inoculum. In the formerculture, that yeast extractin the mediumtends to cause this type of growthpersisted for the durationof friability; 2,4-D acts to preventthe tendency the experiment; in the latter,the friable condi- towardfriability. At a given 2,4-D concentration, tion has arisen. A rough measureof the friable e.g., 10-5M, increasingthe yeast extractconcen- conditioncan be made by countingthe numberof trationfrom 1 to 5 g./l. producesa markedchange pieces per flaskat the end of the cultureperiod. fromthe RS typeto the friablecondition. There In fig. 6 is illustrateda callus cultureproduced is some evidence to suggestthat friable tissues, April,1957] TORREY AND SHIGEMURA-GROWTH IN CALLUS 339

1000 10-6 M o 0~~~~~~~~~~~~~~~~~5 WETWEIGHT DRY WEIGHT

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TIME IN WEEKS

AVERAGE INCREASE 20 AVERAGE NUMBER

IN WET WEIGHT OF PIECES

a PER FLASK(MG) I S PER FLASK

600

Fig. F Fi. 9. (Tp) The verage eight pr cultue in mg of pearoot calus tisue liqidmed10grow in

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Fig. 9-10.-Fig. 9. (Top) The average weight per culture in mg. of pea root callus tissue grown in liquid media containing 1 g./l. yeast extract in the presence of differentconcentrations of 2,4-D.-Fig. 9a (left) shows wet weights; initial wet weight was 90 mg.-Fig. 9b (right) shows dry weights; initial dry weight was 11 mg.-Fig. 10. (Bottom) A comparison of the average wet weight increase of pea root callus tissue grown in differentliquid media for 8 weeks (fig. lOa, left) with the friabilityof the cultures as measured by the average numberof callus pieces per culture (fig. lOb, right). Horizontal coordinates of the two figuresare the same. 340 AMERICAN JOURNAL OF BOTANY [Vol.44 once formed,will tolerateand actually be stimu- callus tissue,does, in fact,possess organised root- lated in their growth (wet weightincrease) by like structuresis evidentfrom the followingex- higher yeast extractconcentrations than will RS periment. Small separate pieces of the friable callus tissues. callus tissue were transferredfrom medium in The anatomicalbasis for differencesin the RS which theyhad developed (5 g./l. yeast extract and friabletype of callus.-In an attemptto dis- plus 10-5M 2,4-D) to flaskscontaining either the cern anatomicaldifferences between the two types M6 medium(low yeastextract plus 2,4-D) or the of tissueswhich might help to explain theirdiffer- original modifiedBonner medium,lacking both ence in behaviour,histological preparations were yeast extractand 2,4-D. The lattermedium had made of the two distinctivetissues. The callus been shownto be optimumfor normalroot devel- pieces werefixed with aspiration in formalin-aceticopment of excised root tips. In the M6 medium, acid-alcoholfixative, were dehydratedin an ethyl- the tissuecontinued to growin the same partially butyl alcohol series, embedded in "Tissue-Mat" organizedmanner characteristic of the friablecal- and sectionedon a rotarymicrotome at 8,u. Sec- lus. In the Bonnermedium, however, the nodular, tions were stainedwith Heidenhain'shematoxylin partiallyorganized structures present in thefriable and safranin. tissuesdeveloped into normal,well-organized roots In the photomicrographof a sectionof an RS (fig. 7) each projectingfrom the tissue in the callus (fig. 11) is evidentthe unorganized arrange- same orientationas the nodular structurefrom mentof cells characterizingthe growthof the RS whichit was derived. type of callus. The callus piece increasedin size The formationof cell suspensionsfrom friable as a sphere by randomunoriented cell divisions callus cultures.-Callus tissueof the friabletype, with irregularcellular enlargementand differen- whencontinued for prolonged periods with regular tiation. The lack of organizeddevelopment is evi- additionof freshmedium to the flask,produces a dentthroughout the tissuemass includingthe peri- cultureof many hundredsof small-tissuepieces pheral tissuesexposed directlyto the medium. In and the culture medium itself becomes a dense fig. 13 is shown at high magnificationa region suspensionof isolated viable cells. The cell sus- selectedat randomfrom the RS typecallus where pension may representa considerableproportion the lack of organizationis clearly evident. The of the weightof theculture i.e., up to 20 per cent tissue is compactand dense with a few isolated of the freshweight. In fig.14 is representedunder scatteredareas of tracheidswhich show secondary high magnificationa sample of this cellular sus- thickeningand pittedwalls. pension. The cells in the mediumvary in size The histologicalappearance of the friabletype from20,t up to over lOOu.in certaincases. De- callus tissueis quite different.In fig.12 is seen a pendingon the age of the culture,a large propor- sectionfrom a callus originallygrown as the RS tion of t,hecells appears to be viable with cells type and then transferredto a mediumof high usually showinglarge vacuoles criss-crossedwith yeast extract concentrationwith a subsequent cytoplasmicstrands in whichprotoplasmic stream- change in anatomicalcharacter to the friabletype ing is clearly evident. The nucleus may be sur- of growthat the peripheryof the tissue. The re- rounded by starch grains and other particulate gion of friable tissue is most strikinglydifferent structures may be presentin the cytoplasm.Usu- in the degree of its organization.Instead of the ally the cell wall is evidentand is readilydemon- homogeneousunorganized tissue evident in fig.11, stratedby simple plasmolysisof the protoplast. the friable tissue shows primordium-likeareas of Some plasmolyzedcells can be discernedin fig.14. organizationwith extensiverows of cell lineages No conclusiveevidence has been gained that such and structuresreminiscent of rootapical meristems cells actually divide after they have been sepa- and root caps. Betweenthese organizedstructures rated fromthe tissue mass fromwhich they were are regions of sloughed-offcells which serve to derived. separatethe peripheraltissue into discretenodular Any explanationof the friable conditionand structuressome of which dissociateand fall into the separationof cells mustultimately rest at the the nutrient medium. These separated tissue biochemicallevel. Analysisof the basis for fria- masses continuecell divisionand enlargementin bilityhas been hamperedby the complexityof the a shnilar, partially organized manner. Thus it medium. Yeast extractis a complex mixtureof appears thatthe. major differencein behaviourof organic and inorganicmaterials and its presence thetwo tissuetypes resides in the degreeof organi- in the mediumin quantityis not conduciveto a zation. In the presenceof high yeastextract con- ready clarificationof the problem. Work is thus centrationin the medium relative to the auxin proceedingtoward the developmentof a synthetic concentration,the callus tissue shows a marked mediumwhich will allow manipulationof the nu- tendencytoward organization.This tendencyto- tritionof the tissue under more carefullycon- ward organ formationis suppressedby high 2,4-D trolledconditions. concentrationrelative to the level of yeast extract DIsCUSSION.-The appearance of morphologi- in the medium. cally distinct tissue forms in callus cultures has That the friable callus tissue, unlike the RS been repeatedly reported in the literature (Gau- April,19571 TORREY AND SHIGEMURA-GROWTH IN CALLUS 341 theret, 1955a; Henderson, 1954; Nobecourt, trumof organizationin a geneticallyhomogeneous 1955; Reinert 1956; Tryon,1956). In agar cul- tissuesystem is subjectto studyand analysis. ture these differencestend to be suppressedand That 2,4-D suppressesorganization in thistissue oftenhave been disregardedby tissueculture work- systemseems clear. This behavior of 2A4-Dis ers, since the tissue pieces, althoughof strikingly analogous in certainrespects to that describedby differentappearance, are known to have had a Skoog (1954) for indoleaceticacid in tobacco commonorigin from a particularcallus strain. In stem segments.The suppressionof organ forma- his review Gautheret(1955b) has discussedcer- tion can be reversedby increasedconcentrations tain modificationsof this sort which have been of yeast extract.Further evidence concerning the natureof the yeast studied physiologically.They have been shown extracteffect depends upon the establishmentof a syntheticmedium capable of to be due to distinct for physiologicalchanges as, maintainingthe pea root callus tissue in continu- example, in the tis- "anergized" (or habituated) ous culture. It seemsclear fromthe presentwork sues whichno longer requireauxin in the medium thatthe nutrientrequirements for continued and are meri- morphologicallydistinct from the original stematicactivity of the rootvascular cambium and tissues. The cause of thesetissue modificationsis its callus derivativesare greaterthan thoseof the not at all certain,although in the case of auxin apical meristemor forthe initiationof lateral root anergyit is believed to be a kind of enzymatic meristems.How complex these requirementsare adaptation (Gautheret,1955b). Such a modifica- remainsto be determined.It is also a matterof tion may arise in the tissuespontaneously and un- interestto determinethe relationshipbetween the controllablywithout deliberate of the manipulation requirementsin vitroand those in vivo. Clearly, externalnutrient medium. 2,4-D is an extraneousstimulator of cell division, In certainother cases, thereare reportsof mor- butmay be replacedin theplant by an auxinwhich phologicallydistinct modifications which are at- acts togetherwith other factors supplied in the root tributabledirectly to manipulationof the nutrient to initiatethe vascular cambiumat a particular medium. The best documentedcase to date is that locus withinthe plant body. workedout by Skoog and his associates (Miller Several reportsof the complexinteraction in the and Skoog, 1953; Skoog, 1954) who have shown growthof callus tissueof auxinsof the 2,4-D type tobacco pith callus tissue to respond in distinct and a complex nutrientare of interest.Steward morphogeneticpatterns to manipulationof the and Caplin (1951) and Steward and Shantz nutrientmedium, especially of certaincomponents: (1956) have reportedthe synergisticinteraction auxin (indoleaceticacid), adenine (or kinetin), of 2,4-D and-coconut milk in the growthof potato phosphateand amino acids. In the tobacco stem tuber callus tissue with a furtherstimulus to segmenttest of Skoog, increasingauxin concen- weight productionby casein hydrolysate.These trationat a constantadenine level caused increas- authorsreported no effectof variationof the com- ing inhibitionof bud formation,whereas at an ponentsof the mediumon organogenesisin the optimumlevel of auxin, root initiationwas pro- potato tissues, but did indicate differentialre- moted. In mostof thesestudies on tobaccotissue, sponses in termsof cell divisionversus cell en- Skoog did not use clonal callus tissueswhich had largementdepending upon the structureof the been previouslymaintained as essentiallyundif- auxin used. In potatotuber tissue, 2,4-D appears ferentiatedtissues. His work does show,however, to stimulatecell division and enlargementabout the importanceof the nutrientmedium in affecting equally. The active componentsof the medium the morphologicalexpression of the tissuesin cul- remain to be completelydetermined. Jagendorf ture. (1952) reportedthe synergisticinteraction of p- The pea root callus tissue describedhere is of chlorophenoxyaceticacid and yeast extract in particularinterest since it representsa tissue sys- "tumor" formationin cabbage roots. The effect tem in whichthe degreeof differentiationand/or of the ye.astextract could be replaced by organic organogenesiscan be controlledin culturedroot nitrogenin the formof urea, asparagineor simply tissuesby manipulationof the componentsof the by inorganicnitrogen as ammoniumchloride. In nutrientmedium. Such a systemoffers a meansof thiswork, studies were made of seedlingroots and studyingthe factorscontrolling the expressionof the tumorswere formedon the roots ratherthan organogenicpotentialities in roots. Root callus on isolated callus tissues. Jagendorfand Bonner tissuemaintained in an unorganizedcondition for (1953) were able, however,to establishand main- many monthsthrough numerous subtransfers can tain such callus tissuein vitroon the same media. be made suddenlyto formnormal, organized root No effectsof thedifferent media on themorphology structuresby changingthe constituentsof the nu- of the callus tissues in culture were described. trientmedium. Further,all intergradationsof or- Tryon (1955) reportedthe formationof tumorous ganogenesisfrom completely organized root struc- growthson seedlingroots of tobacco grownin a turesdown to completelydisorganized cell suspen- medium containingmalt and yeast extract. She sions can be producedat will by manipulationof has been able to culture these callus tissues in the componentsof the medium. Thus, the spec- 'vitro.Nickell (1955) has establishedcallus tissues 342 AMERICAN JOURNAL OF BOTANY [Vol.44 April,1957] TORREY AND SHIGEMURA-GROWTH IN CALLUS 343 fromroots of thesweet clover, Melilotus officinalis, RS type with commercialpectinase preparations using Jagendorf'stechnique. using very small tissue pieces has been found Reinert (1956) has recentlydescribed another possiblewith the methodof Chayen(1952), which case in which change in the constituentsof the pointsto pecticcompounds as the criticalbinding medium has resulted in strikingmorphological agents in the root callus tissues. It is suggestive modifications,gi^ving rise to extremelyfriable tis- thatthe conditionsleading to partial organization sue. Omission of certain componentsfrom the of the callus tissue, e.g., the formationof pre- nutrientmedium is suggestedas a possible cause sumptiveroot primordia,are the conditionsunder of the modificationin growthform in spruce which friabilitybegins to appear. It is possible tissues,Picea glauca. Still otherreports indicate thatchanges in pecticsubstances usually associated that the nutrientconditions may affectthe mor- withthe dissolutionof the middle lamellae in the phologyof the tissue cultures,giving rise to ex- sloughingof root cap cells in an organizedroot treme friability. Muir et al. (1954) reported meristemoccur also under the particularnutrient growingseveral tissues in the presenceof yeast conditionswhich lead to friabilityin culture. If extractwith constant agitation, leading to the pro- such is the case, these changesare suppressedby duction of cell suspensions. Similarly, Steward the presenceof 2,4-D. No directevidence for this and Shantz (1956) describe a liquid culture possibilityhas been elicitedin the presentwork. methodinvolving large numbersof carrottissue Unfortunately,little is knownabout the chemistry explantswhich during culture with constant agita- of the rootcell wall componentsor of the enzymes tion, producefree floatingcells. In this case the involvedin theirmetabolism. friabilityis perhaps associatedwith the changing conditionof the nutrientmedium following peri- SUMMARY ods of culturewith large amountsof tissue. Excised root tips from germinatedpea seeds, In the pea root callus culturesdescribed here, variety Alaska, were induced to form vascular the friablecondition is attributabledirectly to the cambia by treatmentwith IAA. Tissues isolated constitutionof the nutrientmedium. Even on agar from the vascular cambiumregion of such root medium,the friable conditionoccurs when the tips have been grownas a root callus tissueon a mediumcontains high levels of yeast extractrela- nutrientmedium containing the componentsof a tive to the 2,4-D concentration.With constant modifiedBonner medium: i.e., major salts, vita- agitationthis friabilitybecomes most evidentand minsand 4 per centsucrose and, in addition,pow- the resultingsuspension of innumerableviable dered yeast extractand 2,4-D. The clone of pea cells of higherplant tissue is a remarkableprod- root callus tissue thus establishedwas shown to uct which should prove of considerableexperi- require both the yeast extractand the auxin for mentaluse. increasein weightwith an optimumconcentration Any explanationof the friable conditionmust of 1 g./l. yeast extractand 10-6M 2,4.D. The involve the physiologyand chemistryof the cell callus grew on a solidifiedagar mediumor in walls or the middle lamellar materials which liquid flask culture if constantlyagitated. In cementthe cells together.The fallingapart of the liquid culturetwo morphologicallydistinct tissue tissue is probably due to breakdownof calcium formswere producedin responseto differentrela- pectate, the cementingmaterial of the middle tive concentrationsof yeast extractand 2,4-D in lamellae. Such a breakdowncould occur either the medium.A solid, round,smooth type of callus throughenzymatic action, e.g., a pectic enzyme tissue produced by essentiallyunorganized tissue capable of dissolvingthe pectic materials,or by growth(RS type) was formedat optimumcon- removal of the calcium by chelationwith a re- centrationsfor productionof increase of tissue sultantloss of the cementingproperties of the mid- weight. At high yeast extractconcentrations rela- dle lamellae. The presentexperiments do not give tive to the 2,4-D concentration,an extremelyfri- the evidencenecessary to decide the issue. Thus able type of tissue was producedwhich resulted far, preliminaryexperiments do not supportthe in cultures consistingof many small nodular idea of calciumchelation, although this possibility pieces with heavy suspensions of viable cells in is not to be discarded. Northcraft(1951) has the liquid medium. Anatomicalstudies of the two shownthat externallysupplied chelatorssuch as morphologicaltypes show that in the friabletis- ammoniumoxalate may be used-to cause disinte- sues, partial organizationof root meristem-like grationof higherplant tissues. structureshad occurred. These organizedstruc- Macerationof the compactcallus tissuesof the tureswere shownto be capable of developingas

Fig. 11-14.-Fig. 11. Photomicrographof a section of pea root callus tissue of the RS type showingthe homogeneous unorganized nature of the tissue mass. X 70.-Fig. 12. Photomicrographof a section of pea root callus tissue of the friable type showing on the peripheryof the callus to the right the partial organizationof the tissue into primordium- like structures. Note the alignment of cells in rows and the discontinuitiesbetween primordia. X 70.-Fig. 13. En- larged section fromfig. 11 showing at high magnificationthe lack of organizationof the RS type callus tissue. X270.- Fig. 14. Photomicrographof a sample of cell suspensiontaken fromthe liquid mediumof a friablecallus culture. X 140. 344 AMERICAN JOURNAL OF BOTANY [Vol.44

roots in auxin-freemedium. In the presenceof possibilitiesfor study of root morphogenesisof- yeast extract,2,4-D suppressesorgan formation, fered by the pea root callus which shows such leading to the unorganizeddevelopment character- morphologicaldiversity in. responseto changesin istic of the RS type of callus. Once a tissue has the nutrientmedium are discussed. changedto the friabletype, it has not been pos- DEPARTMENT OF BOTANY, sible to date to growit underconditions in which UNIVERSITY OF CALIFORNIA, it will revertto the original RS condition. The BERKELEY 4, CALIFORNIA

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