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AUXIN-DEPENDENTGROWTHOF EXCISED HELIANTHUSTUBEROSUSTISSUES. I.

'

J. P. Nitschand ColetteNitsch

THIS PAPER is the firstof a seriesreporting our thisresponseis linkedto a comparableincreasein


experiments on the proliferation of Jerusalemarti- respiration.In thefieldof tissueculture,Jerusalem
choketubertissuesin sterileculture. The present artichokehas beenused to obtainpermanent strains
articledeals withtissueculturetechniquesand with (Gautheret,1941; Nobecourt,1941), to testvari-
the effectof mineralsalts, certaincarbohydrates ous compounds(Steward and Caplin, 1952; and
and variousnaturalauxins on the growthresponse manyothers),and to studycertainaspectsof me-
of thesetissues. tabolism. Gautheret,especially,has taken advan-
The vascular parenchymaof the tuber of tage of this materialextensively.He has shown
Jerusalemartichoke(HelianthulstuberosusL.) is thatit respondsreadilyto auxins in tissueculture
an almostideal materialto studygrowthas an inte- (1942a) and he has also publisheda detailedana-
gratedwhole in whichcell divisionas well as cell tomicalstudyof the proliferations occurringas a
enlargement take a part. In thistissue,bothproc- responseto auxins (1953). In the latterinvestiga-
esses can be induced by a single substance,an tion,he observedthattheisolated,roundedcenters
auxin such as indole-3-acetic acid (IAA). Thus, of cell division,suchas theone visibleat thetop of
if we place asepticallya small cylinderof tissue fig.IF, arise aroundsecretory canals. He also con-
takenfroma maturetuberon a mediumcontaining cluded that both pith and xylemparenchymare-
sugar and mineral salts, very little increase in spondsimilarlyto auxins.
weightoccurs and cell divisionis certainlyat a STUDY OF THE TECHNIQUES.-Plant material.-
minimum.The additionof a very small amount The strainsof plant tissueswhichhave been iso-
of IAA to themediumstartsan abundantprolifer- lated by variousworkersin the fieldof tissuecul-
ationwhichis apparentin thenewcellsthatcan be ture are generallyheterogeneous,both morpho-
seen under the microscope (fig. 1) and in the logicallyand physiologically.In addition,aftera
increasein freshweight(fig. 2). The increasein certaintimeof cultureand underthe influenceof
freshweightis almostdirectlyproportionalto the some as yet ill-definedfactors,the propertiesof
logarithm of theauxin concentration up to a maxi- these strains change. Such a change has been
mumbeyondwhichgrowthdecreases (fig. 2, 16- illustratedin the so-called"habituation"to auxin
18). The anatomicalexaminationshowsa behavior observedby Gautheretand others.The habituated
which differsaccordingto the concentrationof strains,in turn,may revertto auxin sensitivity
IAA. Whenthe auxin level is around5 X 10-8M, (Nitschand Nitsch,1955). Thus,mostofthestrains
cell divisionis orderly,and a cambiumlikelayer whichhave been subcultured repeatedly are nottoo
becomesvisibleat a certaindistancefromthe per- well suited for reproduciblephysiologicalwork
ipheryof the explants (fig. IC, 1D)). At higher (Caplin, 1947; Gautheret,1955a). This state of
auxin concentrations-around 5 X 10-6M for ex- affairshas been well understoodby Caplin and
ample-cell division proceeds also from isolated Steward(1948) whodecidedto use alwaysexplants
centers distributedthroughoutthe mass of the fromthe originaltissue,in theircase, the carrot
tissue (fig. IE, IF), resultingin the randomtype root. This has been successful,althoughwiththe
of proliferation seen by otherworkers. complicationthat individual carrots were found
The tuberof Jerusalemartichokehas been used laterto differsomewhatin theirresponseto coco-
extensivelyby many botanistsas a materialre- nut milk fractions(Steward and Shantz, 1954).
spondingwell to various auxins. Reinders(1942) A clonal materialcould possiblybe less variable.
observedthat IAA caused a large increasein the From the Etablissements Vilmorin,in France,the
water uptake of Jerusalemartichoketuber disks. threevarietiesof Jerusalem artichoke, "Blanc Com-
Hackett and Thimann (1952) have shown that mun,""Patate" and "Piedallu 17" were obtained.
To makesurethatwe wouldbe dealingwithgenu-
1 Received for publication April 12, 1956. This work ine clones,we have selectedone strainwithineach
was startedin 1952 at the Centre National de Recherches
Agronomiques,Station de Physiologie Vegetale, Versailles, of thesethreevarietiesby growinga stockin the
France. It was continued at Harvard Universityin Pro- fieldfroma singletuber. Fromthenon, thestrains
fessor R. H. Wetmore's laboratory,supported in part by were always propagatedvegetatively.The tubers
a grantmade to ProfessorR. H. Wetmoreby the American were harvestedin the fall beforefrostand stored
Cancer Society, Inc., as recommendedby the committee
on Growthof the National Research Council, and in part in sand in a cold room at 0-50C. The sensitivity
by a grant-in-aidmade by the National Science Foundation to IAA of thesestrainswas checked(fig. 2). The
to ProfessorsR. H. Wetmore and K. V. Thimann. selection"P17" was somewhatmore sensitiveto
The authors would like to express their gratitude to auxin than the two othersand was used through-
ProfessorR. H. Wetmore and also to Dr. Georges Morel
for the facilities and the kind help which they so gener- out thereportedexperiments.
ously provided. The explant.-The standardization of the initial
839
840 AMERICAN JOURNAL OF BOTANY [Vol. 43
Deceomber,1956] NITSCH AND NITSCH-GROWTH OF EXCISED TISSUES 841

explant is importantbecause (1) the relative carded. This methodeliminatesthe artifactsap-


growthWt- Wo, in which Wt is the final and pearingwhenthe same culturesare replantedafter
Wo the initialweight,varies withthe size of the weighing,a procedurewhichincreasesthe growth
inoculum (Caplin, 1947; Duhamet, 1955) and of the replantedculturesover that of the undis-
because (2) it saves weighingthe culturesat the turbedones,as shownby White(1953). The results
beginningof theexperiment. Experiments in which (fig. 3) indicate that the fresh weightof such-
the size and the shape of the initialexplantwere culturesfollowsa sigmoid curve, the maximum
variedindicatedthatcylindersof tissueabout 2 X growthrate occurringapproximatelyduring the
3 mm.and weighingfrom15 to 22 mg. grewwell second week of culture. This patternresembles
and uniformly, Pearson'scoefficientof variationof thatobservedby Caplin and Steward (1945) with
the mean increment in frestweightbeing generally liquid media supplementedwith coconut milk.
8-12 per cent. These results,as well as the anatomicalobserva-
When plant tissues are grown in a stationary tions, indicated that a 3-weekperiod was long
manner,it is important to considerthe polarityof enoughto give an adequatepictureof the growth.
the tissuessince, as shownby Gautheret(1944), promotingcapacityof the medium. Accordingly,
theapical and thebasal endsof theexplantrespond the cultureswere grownfor a period of 3 weeks-
differently. In our experiments, the explantswere throughout thisinvestigation.
obtainedas follows:a radial cylinderwas removed The effectof agar.-Steward et al. (1952) have
fromthetuber;thiswas cutintocirculardiskswith shownthat,in theirsystem,rotatedliquid media
tangentialfaces. Each disk was laid down,one of cause a muchgreaterincreasein freshweightthan
its flatfaces (taken at random) being in contact stationarymedia solidifiedwith agar. On the
withtheagar. In thismanner,boththe apical and otherhand, Heller (1951) has demonstrated that
the basal ends of the explantwere always in con- the physicalcontactof a plant tissue with a col-
tactwiththe medium. loidal structure, suchas thatprovidedby agar gels,
Finally, since a preliminarysoaking in water causes a muchbettergrowththanthe contactwith
increasesthe responseto auxin of grass coleoptiles a liquid solution.Because of the lack of adequate
(Nitschand Nitsch,1956) theeffect of washingthe facilities,we had to use the old methodof solidify-
explants before plantingwas investigated.This ing our media withagar. This was a disadvantage
pre-treatment did nothave any significantinfluence sinceagar containsmineralsalts (see table 1), and
on the responseto IAA, however,so that the ex- also vitaminssuch as thiamin (Day, 1942) and
plants were not washed before plantingin the biotin (Robbins and Ma, 1941). The purpose of
presentexperiments. thisinvestigation beingto studytheeffect of auxins,
In summary,the procedurefor the preparation it was important to ascertainthatagar was devoid
of theexplantwas as follows.A healthyJerusalem of such substances.It was foundthat a complete
artichokewas washed, peeled and sterilizedby medium(withsugarand mineralsalts) but lacking
immersioninto a 7 per cent (w./v.) filtered"Pitt- added auxin, solidifiedwith 1 per cent unwashed
chlor"solution(a commercialpreparationcontain- Difco "Bacto-agar,"did not cause any of the cell
ing 70 per centcalciumhypochlorite)2. It was then
placed betweensheetsof sterilepaper towelsand TABLE 1. Typical mineral analysisa of agar
bored throughradiallywith a stainlesssteel cork
-- 0.15 per cent
borerof 3 mm.diameter.The ends of the cylinder Nitrogen --

of tissuethusobtainedwerediscardedand the rest Ash ---- 3.5


cut to size withtwoparallelrazorblades distantof Na.O + K20-- 0.2
about 2 mm. MgO --- 0.4
CaO ---0.8
The effectof timeon growth.-To decide when
Fe2O3 + A1203.- - 0.04
the culturesshouldbe harvested,one shouldknow MnO ---- 0.01
the timecourse of growth.Therefore,four dozen 0.9
Si ,2 -------------------------------
cultureswerestartedat thesame time. Fromthese, S03 --- 1.03
a sample of twelvecultures,harvestedat random, P205 --- 0.01
was weighedafter4, 7, 14 and 21 days, and dis- CO2 --- 0.03
C --- 0.01
2 Manufacturedby the Columbia-Southern
Chemical
22, Penn.
Corp.,Pittsburg a Detroit,Michigan.
CourtesyDifco Laboratories,

Fig. 1. Anatomical changes induced by various concentrationsof indole-3-aceticacid in Jerusalem artichoke tuber-
tissues grownfor 21 days on mineral salts (M2 + minor elements), 5% sucrose, and 1% agar. In this particular case,
the cylindricalexplants had been laid down on the mediumon their tangentialfaces. The cultureswere sectioned at
rightangles to the longitudinalaxis of the original cylinder,whichis also at rightangles to the surfaceof the media. The
flattenedareas, visible in photographsC and E, occurred at the contact of the cylinderswith the media.-A: no IAA
added.-B: magnificationof section A.-C: 5 X 1O-8M IAA + Na acetate 10-4M.-D: magnificationof section C.-
E: 5 X 10-6M IAA + Na acetate 10-M.-F: magnificationof section E.
842 AMERICAN JOURNAL OF BOTANY [Vol. 43

divisionswhich auxins generallytriggeroff (fig. De Capite (1955) reportedthatcontinuousarti-


1A, 1B) nor stimulateany increasein freshweight ficiallightmarkedly increasedthegrowthof strains
(fig.2, 19). Thus,forpracticalpurposes,the agar of sunflower, Bostonivyand carrotoverthatof the
used did not contributeenoughauxins,if any, to dark controls. Table 2 gives preliminaryresults
interfere withour study. obtained with Jerusalemartichoketissues. They
Although agar does not seem to contribute indicatethatthekindof lightused and thetimeat
auxins to the medium,its concentration influences whichthelightis appliedmayinfluence the growth
thegrowthof the cultures.A studyof the optimal response. In the firstexperimentthe culturesin
agar concentration was made withunwashedDifco thelightbecamegreen,but grewless thanthe ones
"Bacto-agar," a specially purified agar3, and in the dark. It is possible that light inactivated
shreddedagar washed for 16 hr. in runningtap part of the IAA (100 tsg./l.)put in the medium,
water. The pH was 5.5 in each case beforeauto- beforethe culturescould take it up, thusreducing
clavingand theexplantsweresimplylaid down on growthindirectly.In the second experiment, the
top of the media. At the 0.25 per cent agar con- greatestamountof growthwas obtainedunder a
centration, the culturessank somewhatdeeperinto 17-hr.day of continuous"far red" light,but this
thesemi-solidmassthantheydid at the 1 or 2 per- treatment also increasedsomewhatthe temperature
centlevels.In general,thebestgrowthwas obtained of the cultures.The poorestgrowthwas obtained
at concentrations around1 per cent (fig.4A). In a undera 17-hr.day of continuousred light.
different experiment, in which 1 per cent concen-
trationsof shreddedand Difco "Bacto-agar"were
TABLE 2. Effectof light on the growthof Jerusalemarti-
directlycompared,unwashedshreddedagar (fig. choke tuber tissues
4B) was inferiorto washedshreddedagar which,
in turn,was inferiorto unwashedDifco "Bacto- Experiment1: cultures exposed to light immediatelyafter
agar." Accordingly, unwashedDifco "Bacto-agar," planting:
at theconcentration of 1 per cent (w./v.) was used Growthin darkness: (freshweight) 126 mg.
throughout this investigation. Growthin white light (200 ft.-c. continuous
The importance of pH.-It is wellknownthatthe illuminationwith a mixture of fluorescent
pH has a markedeffectupon the reponseof plants and incandescentlight): 91 mg.
to IAA (Thimannand Schneider,1938; Bonner,
Experiment2: culturesleftfor 3 days in darknessimmedi-
1938; etc.), upon the stabilityof IAA in solution ately afterplanting and prior to the exposure to light:
(Kogl et al., 1934), upon theprecipitation of phos- Growthin darkness: 183 mg.
phates and heavy metal salts, the gelificationof
Growth in 17-hr. days of fluorescentwhite
agar, the destructionby autoclavingof organic light: 188 mg.
compounds such as thiamin, pantothenicacid
Growthin 17-hr. days of far red (about 720
(Frost and McIntire,1944), cysteine,tryptophane, msu) light: 217 mg.
etc. For all thesereasons,the pH was adjustedto
Growthin 17-hr. days of red (about 650 mit)
5.5 in all cases beforeautoclaving,a value found light: 168 mg.
optimalin thecase of normaltissuesof Helianthus
annuusL. (Henderson,1954).
Preparationof the media.-The waterused was The expressionof growth.-In our cultures,
double glass-distilledwater. About 20 cc. of instancesof both cell multiplication and cell en-
medium were poured into each 150 X 25 mm. largementcan be found. To get an idea of the
"Pyrex" tube. The tubes were closed with non- overallgrowthachievedduringa given period of
absorbentcotton and covered with polyethylenetime,one may measurethe increasein wet weight,
filmto preventdessication.In a few experiments as has been done by practicallyall the workersin
done later,Kimble screw-capped tubes were used. this field. In the comparisonof two different
These cut downhandlingand ensuregood aeration experiments reported, say,by twodifferent authors,
and sterility(Shunk and Johnson,1941). the value of the gross increase in weightis not
The tubes containingthe media were generally sufficient and this fortwo reasons: (1) obviously,
sterilizedby autoclavingfor20 min.at 15 lb. pres- this increase in weight is dependentupon the
sure. Whenlabile substanceswereused,theywere numberof cells whichwere presentin the initial
sterilizedby filtration through"Millipore" filters inoculum; and (2) it has been shown experi-
whichwe have adaptedfor this purpose.4 mentallythat the size of the initialexplanthas a
Light and temperature.-The temperatureat great influenceupon the results (Caplin, 1947;
whichthe cultureswere grownwas around 25?C. Duhamet,1955). Thus, the size of the inoculum
day and night. This is the temperature whichhas has to be takeninto considerationin the finalre-
been found optimalfor culturesof othertissues sults,contrarilyto Heiler's views (1953) and to
in thedark (De Capite,1955). his exclusive use of the absolute increase in
3 Courtesy of the Difco Laboratories, Detroit,Michigan. freshweight. Let WObe the initial freshweight
4 See Part II of this series. of the explantand Wt that of the cultureaftera
December,1956] NITSCH AND NITSCH-GROWTH OF EXCISED TISSUES 843

F~~~~~~~~~~~~~~~~~~~~~

/3
LX 2s
coLL~~~~~~~/ /L
.. . b) o

z 50 .PC
O s ~~~~~~~~~~~~~100
_~~~ 4 ~~~ 4 2

50-

IL /1 = i L~-

0 I ~~0 ~
9~ 900
IAA CONCENTRATION
(I/A)
o 4 7 14 21
I....- I I I I ~~~~~~~~~~~~~~~T
IME IN DAYS

w
iLC CN A xO
=o W^* , 2 3 4 5____
UD~~~~~~~U
I-0
cn~~~~~~~~~~~~~~~~~~0
chk clueof2mgintawegtMieasouinM+5%ucros +0 IA 5X1-M %aa.Fg

z
us~~~~~~~~

0
w
"Bcoaa" UDS= nahd pcal uiidDfoaa;W wse heddaa.Iiilfehwihso

exlnsoTon
100N 5m. U,US,ad22m.(S.Fg 4B: US 100w/. 1.:
nase heddgr S=1
-0-5 0 20 40 so
I. 2 3 NaCL CONCENTRATION
(MxIO_)
AGAR CONCENTRATION(%

Fig. 2-5.-Fig. 2. Effectof various concentrationsof indole-3-aceticacid upon the growthof tuber tissues of three
clones of Jerusalemartichoke,namely "Patate" (Pa), "Blanc commun" (BC), and "Pi6'dallu 17" (P17). Initial fresh
weight of all explants: 18 mg. Harvest after21 days of culture.-Fig. 3. Growth curve of undisturbedJerusalem arti-
choke cultures of 20 mg. initial weight. Mineral solution M2 + 5% sucrose ? IAA (5 X 10-7M) ? 1% agar.-Fig.
4. Effectof the agar concentrationon the growthof Jerusalemartichoke cultures.-Fig. 4A: UD = unwashed Difco
"Bacto-agar"; UDS = unwashed, specially purifiedDifco agar; WVS= washed shredded agar. Initial freshweights of
explants: 15 mg. (UD, UDS), and 22 mg. (WVS).-Fig. 4B: US = 1% (w./v.) unwashed shredded agar; WVS= 1%
washed shredded agar; UD =1% unwashed Difco "Bacto.agar." Inocula: 22 mg. freshweight.-Fig. 5. Effectof added
Ca(N03)2(0.3 mM) MgSO4 12 (1I mM),
Is KH2PO4 (3 M),sucos (5%), IAA ( 1NX 1- ITM),.agar AI%)Inoc
8414 AMERICAN JOURNAL OF BOTANY [Vol. 43

period of timet. The increasein freshweightis: differingformulaefor mineralmedia, whichindi-


Wt- WO. The resultsshould be expressedas in- cates that each particularstrainmay have some-
creasesin weightrelativeto the initalweight:WO, what different mineralrequirements.No medium
whichmeansthattheyshouldbe expressedas rela- having been studied for the specificpurpose of
growingJerusalemartichoketuber tissues,it be-
tive increasesin weight: t Such a method
-. came necessaryto examinethe nutritiverequire-
wo mentsof thesetissuesin culture.
allows one to state if the cultureshave doubled In doingso, one could use thetriangularmethod
or trebledin weightduringthecourseof theexperi- whichconsistsin maintaining constantthe sum to-
ment. It can be expressedconveniently on a per- tal of mineralions whileexploringall the possible
wt combinations obtainedwhenthe respectiveconcen-
centagebasis: W X 100, the startingpoint trationof theconstituentsis varied. Such a method
wo has been used Hildebrandtet al. (1946), but re-
(when Wt - W.) being represented by 100. Ex- jected by Burkholderand Nickell (1949), and by
pressingthe resultsin this way is legitimateonly
Heller (1953) because it is too rigidand does not
if thetimefactoris constantin all cases, as it was
permitan independent variationof the concentra-
in thepresentinvestigation(all increasesin weight tionof each ion. The approachwe have used here
being recordedafter21 days of culture). Should has been to proceedby successiveapproximations.
the durationof two experiments undercomparison
We have startedwitha mineralsolution,called M1,
then it would be necessaryto intro-
be different,
concoctedfrom the resultsof previous authors.
duce,in addition,thefactortimeand to expressthe we have modifiedthis
Aftera seriesof experiments,
resultsas relativegrowthrates: X - (cf. initialformulato one called M2 (table 3). With
wt t thisformula,the concentration of each individual
Stewardand Caplin,1954). In the presentinvesti- ion was varied over an adequate range. The con-
gation,exceptwhen stated otherwise,the inocula centrationof each ion givingthe best resultwas
alwaysweighed15 mg.,and thedurationof growth noted and servedas a basis for a new improved
in culturewas always21 days. From thesedata it mineral medium,called N1 (table 3) In this
-

is easy to calculatethe relativegrowthrates from medium,again,theconcentration of each individual


the absolute incrementsin freshweighthere re- ion was varied, to check if the concentrations
corded. chosenforN1 werereallygood. This paperwillnot
Statistical significance.-Throughout the work reportthe bulk of this extensiveexamination,but
presentedhere,ten to twelvereplicateswere used onlythe pointsthatare pertinent in demonstrating
foreach concentration of each treatment.In other the effecteach ion has upon the growthof Jeru-
words,each point on any graph is the mean of salemartichoketissues.
ten to twelveindependent replicates.This number The effectofNaCl.-Both Burkholder and Nickell
of replicateswas satisfactoryin our case since (1949), and Heller (1953) have used sodiumand
Pearson's coefficient of variationwas generallyin chlorideas "accompanying ions" becausetheywere
the orderof 10 per cent. Whenthe treatment was withouteffecton the growthof the tissues they
injurious, however,the variabilityincreased,as
alreadyobservedby Heller (1953). TABLE 3. Compositionof the mineral solutionsM2 and N1
THE EFFECT OF INORGANIC SALTS.-Thatcertain
MediumM2 MediumN1
ions,especiallypotassium,increasethe responseof
planttissuesto auxins has been shownby various 1. Salt concentrationin g./l.:
authors,especiallyin the case of oat coleoptiles KNO3 --0.5 2.0
(Thimannand Schneider,1938; Cooil, 1952). In KC --- 0.5 1.5
the fieldof planttissueculture,everyone, of course, NH4NO3 - 0.2
has been faced withthe problemof mineralnutri- MgSO4-7 H2O-0.2 0.25
MgC1,2 -6 H20 -0.2
tion. Strangelyenough,a thoroughstudyof this
KH2PO4 --0.15
aspectofplanttissueculturehas oftenbeenavoided, NaH2PO4-H20- 0.25
as it were. Gautheret(1942b) and Nobecourt CaC12_- _ 0.05 0.025
(1937) simplyused a mineralsolutiondeveloped 2. Ion concentrationin millimoles/l.:
by Knop (1865) forintactplantsand supplemented K -12.7 39.9
it by a traceelementmixtureproposedby Berthelot Na+ -- 1.8
(1934) for micro6rganisms;White (1951), and NH4+- _ _ 2.5 --
Caplin and Steward(1952) used a mineralsolution Mg+ --- 1.79 1.8
originallydevisedby White (1943) forthe culture Ca+ + -- - 0.45 0.23
of isolatedroots. Hildebrandtet al. (1946), Burk- NO3- - -7.4 19.8
holderand Nickell(1949), and Heller (1953) have SO4 ------
------------------------- 0.8 1.0
made detailedstudiesof the mineralnutritionof PO4H2- - 1.1 1.8
certainstrainscultivatedin vitro. They proposed Cl --9.56 0.46
December,1956] NITSCH AND NITSCH-GROWTH OF EXCISED TISSUES 845

workedwith,at leastbelow5 X 10-3/M. We have Nitrateand ammoniumions.-Growthincreased


checkedthisfactin thecase of Jerusalem artichoke markedlywithincreasingconcentratians of NO3-
tissues by adding increasing concentrationsof ions up to about20 mM/l. (fig.10). WhenNH4+
NaCl to a completemediumwithoutsodium.The ions were introducedintothe mediumin the pres-
results(fig. 5) indicatedthat,in the presenceof ence of 10 mM/l. of nitratenitrogen,the well--
potassiumions, NaCl may be slightlystimulatory,knowninhibitory effectof ammoniumions became
but that this effectdoes not change over a wide apparent(fig.11). As littleas 0.1 mM/l. of NH4Cl
range (5 to 10 mM/l.). The use of Na+ and Cl- reducedgrowth.This effectcannotbe ascribedto
ions may thus be regardedas not modifying the pH (whichwas adjustedto 5.5) nor to a chemical
general growthresponse of Jerusalemartichoke denaturation of the sugar presentduringautoclav-
tissues,at leastin thepresenceof K+ ions. In con- ing since the toxic effectsof NH4+ ions remain
sequence,we have used Na+ and Cl- as accom- whenthe media are sterilizedby filtration, accord-
panyingions. ing to Heller (1954) . At any rate, filteredam-
Effectof potassium.-Whenthe potassiumcon- moniumchloridehad a slightstimulatory effectin
centration was variedfrom0 to 120 millimolesper the absenceof nitratesat low concentrations (0.5,
1.,theresultsdepictedin fig.6 wereobserved.It is 1 mM/l.), but became toxic at higherconcentra-
of interestto note that considerablegrowthoc- tions (fig.11, dottedline).
curred with no added potassium. On the other Soiurcesof sulfur.-Agar being the calciumsalt
hand, the sodium present in the medium (24 of the sulfuricester of a polysaccharide(Fair-
mM/l.) may have compensatedpartiallyfor the brotherand Mastin,1923), one mightexpectthat
absenceof added K+ ions. At highpotassiumcon- no added sulfuris necessary.However,the sulfur
centrations,such as 120 mM/l., growthwas re- in the agar moleculemay not be too readilyavail-
duced. The optimumconcentration of K+ ions is able sinceadded S04- ions increasedgrowthsome-
situatedaround40 mM/l. This value was adopted what(fig.12), theoptimalconcentration oscillating
in the finalN1 formula. between1 and 2 mM/l. accordingto the experi-
Calciumand magnesium.-Figure7 givesthe re- ment. Other sulfur compoundssuch as sodium
sults obtainedwiththe mediumN1 in whichcal- hydrosulfite (Na2S204) provedto be very inhibi-
cium and magnesiumhave been omitted.In the tory(fig.12, dottedcurve). Amongthiscategory,
absenceof added Ca+ + and Mg+ +, an appreciable one may place sodium sulfite(Na2SO3), sodium
amountof growthoccurswhichcan be decreased bisulfite (NaHSO3) and sodium metabisulfite
by theadditionof 0.5 mM/lof sodiumoxalateand (Na2S9O5), all of whichcompletely inhibitgrowth
broughtto zero by the additionof 5 mM/l.of the at the concentration of 1 mM of sulfurper 1. (fig.
same salt. Whenno magnesiumions are added to 13). Similarresultswereobtainedwhenthe auxin
the medium,the additionof small amountsof cal- added to themediumwas 1-naphthalene aceticacid.
cium (0.5 mM/1.) increase growthconsiderably, Additionof lmM/l.of Na2SO4 did not changethe
the effectincreasingverylittleupon furtheraddi- degreeof inhibition causedby 1 mM/l.of Na2S204.
tions of calcium. If we now turnto the effectof Autoclavedsodiumsulfide(Na2S) was not inhibi-
added magnesiumin the absenceof added calcium, torybut was not as good as the sulfate. On the
then we observe that1small amounts (0.25-0.5 otherhand,sodiumthiosulfate(Na,S203) was gen-
mM/l.) of added Mg+ + ions decrease growth erallyas good as sodium sulfate. Cysteine,when
somewhat, but thathigherconcentrations are bene- filtered,was inhibitory, but this effectcompletely
ficial (fig. 8). Finally, when calcium and mag- disappearedwhenit was autoclavedwiththemedia
nesiumare added together, a curvesuch as thatof (fig. 13). Nickell and Burckholder(1950), who
fig.8 (dottedline) is obtained. The bulk of our foundthatcysteine was as gooda sourceofsulfuras
experiments showedthat the optimumgrowthoc- Na2SO4fortheculturesof virustumorsof Rumex,
curswhenabout0.25 mM/l.of Ca++ together with
had probablyautoclavedthisaminoacid. It should
about 1 mM/l.of Mg+ + are added to themedium.
also be noted that these authors reportedthat
Phosphates.-When various concentrationsof
monobasicorthophosphate are added to themedium sodium bisulfitecaused excellentgrowthof their
inhibitedgrowthin
N1 (withoutphosphate),a curveof thetvpeshown cultures,whereas,it completely
in fig.9 is obtained.In general,the optimumcon- our experiments.
centrationof NaH2PO4 was foundto be around2 Minor elements.-Underthe conditionsof our
mM/l. Pure sodium tripolyphosphate5 (Na5P3O10) experiments,the additionof iron,manganese,zinc,
and "Calgon" (commercialsodiumhexametaphos- boronand otherminorelements, eitheralone or in
phate (NaPO3)6) had a bettergrowth-promoting a completemixture, never improved growthover
effectthanorthophosphate whenused at an equiva- that of the controlswithoutthese additions. This
lentlevel of phosphorus.Filteringthe sodiumtri- meansprobablythatthe Pyrexglass,the agar and
polyphosphateinstead of autoclavingit did not impuritiesin the salts used providedan adequate
changetheresults. supplyof trace elements,althoughthe chemicals
5 Courtesyof the Monsanto Chemical Co., St. Louis, Mo. used wereof the "Reagent"grade. For thisreason
846 AMERICAN JOURNAL OF BOTANY [Vol. 43

12,150~ ~ ~~
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December,1956] NITSCH AND NITSCH-GROWTH OF EXCISED TISSUES 847

no minorelementwas added to the basal medium TABLE 4. Efect of glucose and fructosewhen autoclaved
N1. with a completemediumin the presence or absence of
THE EFFECT OF CARBOHYDRATES.-The growth sucrose
responseto auxins of many plant tissues is in-
creasedby thepresenceof sugar. Such is the case, (mg.)
Increase in fresh
forexample,of grass coleoptile(Schneider,1938)
Medium weight
and firstinternodesections (Nitsch and Nitsch,
1956). It was of interest, to ascertainif Completemediuma+ 0.1 M sucrose
therefore, 158
the auxin-dependent growthof Jerusalemartichoke Complete medium + 0.1 M sucrose
tissueswas influenced also by the amountof sugar + 0.1 M glucose -232
available. A series of media containingthe com- Complete medium + 0.1 M sucrose
pletemineralsolutionN1plus 5 X 10-7M of IAA + 0.1 M fructose - - 163
withsucroseconcentrations increasingfrom0 to 10 Complete medium + 0.2 M sucrose 156
Complete medium + 0.1 M glucose 188
per cent (w./v.) was preparedand autoclavedas Complete medium + 0.2 M glucose 217
usual (15-20 min. at 15 lb. pressure). (It should Completemedium + 0.1 M fructose 106
be remembered thatautoclavinggivesrise to traces Complete medium + 0.2 M fructose 72
of glucoseand fructose, as observedby Ball, 1953).
The results(fig.14) werestriking.Withoutadded
a Mineral solution N1 + IAA (5 X 10-7M) + agar
(1%); pH adjusted to 5.5.
sucrose,there was practically no growth, and the
increasein weightvaried withthe amountof su-
crose present,the optimumbeing somewhatunder to occurin nature. These naturalauxins are IAA,
8 per centby weight.Whensucrosewas sterilized its nitrile (IAN), its ethyl ester (IAE), and prob-
by filtration,the culturesgrewless. This observa- ably cis-cinnamic acid. A comparisonbetweenthe
tion led us to investigatethe effectof sucrose,glu- auxinshavingan indolenucleusshowedthat on a
cose and fructosesterilizedby filtrationon one molarbasis, IAA and IAE were about equally ac-
hand, and by autoclavingon the otherhand. The tive (fig. 16). On the contrary,as already re-
resultswere striking.When filtered, sucrosegave ported (Nitsch, 1955), IAN was inactive. This
somewhatbettergrowth(at the 0.2 M conc.) than compoundhas been ascribeda slightauxin activity
glucose or fructosealone (fig. 15). Afterauto- by Bouriquet (1954), but it is possible that the
claving,however,all threesugarsused singlygave sampleused by this authorwas impureand, also,
bettergrowththanbefore,but in somewhatdiffer- thatautoclavinghad caused the formation of some
entmanners.Fructosewas thepoorestof thethree IAA. We were fortunate, throughthe kindnessof
and becameinhibitory, as theconcentrationwas in- ProfessorK. V. Thimann,to be able to use a
creasedfrom0.1 to 0.3 M. Sucrosewas betterthan crystalline sampleof IAN ofthehighestpurity, pre-
fructose,but the best of all was glucose which paredin thelaboratoryofProfessorE. R. H. Jones;
caused a large amountof growth,somewhatap- in addition,we sterilizedthe auxins by filtration,
proachingthat obtainedwithvery potentgrowth thusavoidingthe artefacts mentionedabove.
substancessuch as coconutmilk. The possibility The amide of indole acetic acid (IAAm)6 was
thatglucosecould increasegrowthwhen added to active (fig. 17), but less than IAA, the activity
a mediumcontainingalready0.1 M of sucrosewas startingat about 10-6M, whereas it begins at
investigated.The results (table 4) showed that, 10-9M withIAA. IAAm is, therefore, from300 to
whenautoclavedwiththemedium,glucoseadded to 1,000timesless activethanIAA, butis notinactive,
sucrosedoes in factincreasegrowth.This, as yet as is IAN.
unexplained,effectof autoclavingglucosewiththe As far as cis-cinnamic acid6 is concerned,it has
mediumis underinvestigation. a definiteauxin activity(fig. 18). Trans-cinnamic
THE NATUREOF THE AUXIN.-In this study,we 6 We are indebted to ProfessorR. L. Wain for a sample
are mainlyconcernedwith the responseof Jeru- of indole-3-acetemideand to Dr. J. van Oberbeek for a
salem artichoketissuesto auxins whichare known sample of cis-cinnamicacid.

Fig. 6-11.-Fig. 6. Effectof K+ ions on the growthof Jerusalemartichokecultures.KCl was added at increasingcon-
centrationsto the followingmedium,containing (per 1.): NaNO3 (20 mM), MgS04-7 H20 (1 mM), NaH2PO4 (4 mM),
NaH2PO4 (0.13 mM), sucrose (5%), IAA (5 X 10-7M), agar (1%o). Inocula: 22 mg. fresh weight.-Fig. 7. Right:
Effectof Ca+ + ions in the absence of added magnesium. Left: Effectof 5 and 0.5 mM/l. of Na oxalate with no added
calcium nor magnesium.-Fig. 8. Effectof Mg++ ions in the absence of added calcium (solid curve), and in the pres-
ence of 0.25 mM/l. of Ca++ ions (dotted curve).-Fig. 9. Comparative effectsof monobasic orthophosphate(white
circles), sodium tripolyphosphate(black circles), and commercial sodium hexametaphosphate-."Calgon"- (squares)
on the basis of equivalent concentrationsof phosphorus.-Fig. 10. Effectof N03- ions added to a solution containing,
per I.: KCI (20 mM), CaCh2 (0.3 mM), MgSO4 (1 mM), NaH2PO4 (4 mM), Na2HP04 (0.13 mM), sucrose (5%), IAA
(5 X 10-7M), agar (1o%). NaCl was added at decreasingconcentrationsfrom50 to 0 mM/l. to balance the amount of
sodium introduced by NaNO3.-Fig. 11. Solid curve: Effectof NH4+ ions, supplied as NH4Cl and autoclaved, to a
medium containing10 mM/l. of N03- ions. Dottedcurve: Effectof filteredNH4Cl added to a medium devoid of nitrate.
848 AMERICAN JOURNAL OF BOTANY [Vol. 43

150_ I I I 97

o 12 NOS4~ 0o109

O
w E~~~~~~~~~~~~~0C
74~~~~~~~~~~~~~~

w~~~~~~~w
4_
wr
-z
z
Z 50-
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0 1 2 3 4 5 6
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SUCROSE CONCENTRATION(%.) 0

SUGARCONCENTRATION
(MxIO)

Fig.12-15.;-Fig. 12. Comparativeeffectof sodium sulfate (solid line) and sodiumhydrosulfite (dottedline) on the
basis of an eq'uivalentconcentration of sulfur.-Fig.13. Effecton the growthof Jerusalemartichokeculturesof 1 mM
of sulfurper 1. (except in the case of filteredcysteine) when given in the formof various sulfur-containing
compounds.
The numbersgivethe actualincreasein freshweightin percentofthatof thesodiumsulfatecontrols.-Fig.14. Effect
of the concentration of sucroseautoclavedwiththe media. Basal medium:N1L ? IAA (5 X 10-7M) + agar (1%)-
Fig. 15. Effectof sucrose (S), glucose (G), and fructose(F) when filtered(left) or autoclaved (right).

but decreasesthe re- it is necessaryto supplyalso botha sugar (such as


acid not onlyhas no activity,
sponseto IAA, thus givingindicationof an anti- sucrose) and mineralelements(mainlypotassium
auxin effectsuch as that demonstrated by Van and nitrate). This is summarizedby the experi-
Overbeeket al. (1951). mentshown in fig. 19. First of all, a complete
CONCLUSIONs.-Jerusalem artichoketubertissues mediumwith sucrose and mineralsalts does not
respondwell to various auxins but, for this re- supportany growthwithoutauxin (No. 1). Con-
sponse to become apparentas a large amountof versely,IAA withagar alone (No. 2), withagar
growthby bothcell divisionand cell enlargement,plusmineralsalts (No. 3), or withagarplussucrose
December,
1956] NITSCH AND NITSCH-GROWTH OF EXCISED TISSUES 849

16}
2000IAA 2 17
200
w/
// ~~~~-O 150-

IAE/ b-- 10

L A, IA
z U-
IAA
-100 / s lo 50 100'200 | w 500l
/~ ~~ ~ ~ ~ 4
w CNAI AT5 w
So

~50 /
) )0 0- 0
IAN0I

0 ~ ~ ~ .? IO d O4 AUXIN CONCENTRATION
Er ' - "' t ' 1 1
CONCENTRATION 200

10

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150- ~ ~ ~ ~ ~ ~
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1 20- 3i 40- 15 60
ioo- z

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U50 0 010 0 50

CINNAMICACID CONCENTRATION
(x6.75x10-*M)
01
I 2 3 4 5 6
ofvariousconcentrations
Fig. 16-19.-Fig. 16. Effect acid (IAA), its ethylester(IAE), and its nitrile
ofindole-3-acetic
(IAN) uponthegrowth of Jerusalemartichoketubertissues.All theauxinsweresterilized by filtration.-Fig.
17. Effect
acid uponthe growth
of the amideof indole-3-acetic of Jerusalemartichoke tubertissues.Comparison withequal molar
concentrationsof IAA. All auxins sterilizedby filtration.-Fig. 18. Effectof cis- and trans-cinnamicacids upon the
growth ofJerusalem artichoketubertissues.The cis-cinnamic byfiltration,
acid was sterilized thetrans-cinnamicacid was
autoclaved.-Fig.19. The effect on growth of variouscomponents of a successfulmediumforJerusalem artichoketuber
tissues.1 = mineralsolutionM2 + sucrose(5%) + 1% agar,without IAA. 2 = IAA (5 X 10-7M) + 1% agar.3 =
IAA (5 X 10-7M) + M2 + agar. 4 = IAA (5 X 10-7M) + 5% sucrose+ 1% agar. 5 = (1) + IAA (5 X
10-TM). 6 = (5) + thefollowing minorelements(mg./l.): ferriccitrate*5 1120 (10), MnCl2.4 H20 (2.5), 113B03
(2), ZnSO4* 7 H20 (0.05), CoCl2*6 H20 (0.03), CuCl2. 2 1120 (0.015), NaMIoO4. 2 H20 (0.025).
850 AMERICAN JOURNAL OF BOTANY [Vol. 43

alone (No.-4), causes onlylittlegrowth.WhenNo. niques has shownthe effectof the durationof the
1 and No. 2 are combined,however,or No. 3 and experiment, the concentration of the agar, the illu-
No. 4, thatis whenwe puttogether:(1) and auxin, minationof the cultures,the criterionof growth,
(2) sucrose,and (3) mineralsalts, then an ex- etc. The investigation of sodium,po-
of the effects
tremelylarge amountof growthoccurs (No. 5). tassium, calcium and magnesiumions, various
The additionof minorelementsis not beneficial phosphates,sourcesof sulfurand inorganicnitro-
(No. 6). In thesubsequentpapersof thisseries,we gen,minorelementsand certaincarbohydrates re-
will studywhat otherfactorsincreasestillfurther, vealedthatincreasein freshweightat a givenauxin
decrease,or otherwise modifythisbasic responseto level is mainlyinfluenced by the concentration of
the combinationof an auxin,sucroseand mineral (1) potassium,(2) nitrateand (3) carbohydrates.
salts. Otherelementshave a less markedeffect,at least
The mineralsolutionN1 whichhas been devel- in thepresenceof 1 per cent (w./v.) Difco "Bacto-
oped as a resultof the presentstudygives a some- agar." The resultsobtainedwereused to establish
whatgreaterincreasein freshweightthanthatpro- the formulaof a basic mineralmediumwhichal-
posed by Heller (1953). It has also been used in lowsin 21 daysan inoculumof 15 mg.freshweight
this laboratory(with the addition of minor ele- to increaseits weight10 fold. Glucose increased
ments)to maintainsuccessfully overrepeatedtrans- growthfurtherwhen it was autoclavedwith the
fers,strainsof tissuesof Scorzonera hispanica medium,but not whenit was filtered.Amongthe
(normal,"habituated"to auxin, and crown-gall) naturalauxins studied,indole-3-acetic acid and its
and of Parthenocissustricuspidata(normal,"ha- ethylesterwere about equally active. The amide
bituated,"and crown-gall). was activeat concentrations 300-1,000timeshigh-
er; the nitrilewas inactive.Cis-cinnamic acid was
SUMMARY activeat concentrations nearly1,000 timeshigher
thanthat of indole-3-acetic acid, whereasits trans
Tubertissuesof HelianthustuberosusL. grow isomer was inactive by itself and reduced the
in vitroby a combinationof cell divisionand cell growthcausedby IAA.
enlargement underthe influenceof added auxins. DEPARTMENT oF FLORICULTURE AND
The selectionof a clonal materialhas produceda ORNAMENTAL HORTICULTURE,
more uniformtestingmaterialthan that used by CORNELL UNIVERSITY,
manyotherworkers.A detailedstudyof the tech- ITHACA, NEW YORK

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