Professional Documents
Culture Documents
'
J. P. Nitschand ColetteNitsch
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
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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
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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
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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).
16}
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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
LITERATURE CITED
BALL, E. 1953. Hydrolysisof sucrose by autoclavingmedia. metabolites and inhibitors to growth in the Avena
A neglected aspect in the technique of cultureof plant coleoptile. Plant Physiol. 27: 49-69.
tissues. Bull. Torrey Bot. Club 80: 409-411. DAY, D. 1942. Thiamin contentof agar. Bull. Torrey Bot.
BERTHELOT, A. 1934. Nouvelles remarques d'ordre chimique Club 69: 11-20.
sur le choix des milieux de culture naturels et sur la DECAPITE, L. 1955. Action of light and temperatureon
maniere de formulerles milieux synthetiques. Bull. growthof plant tissue cultures in vitro. Amer. Jour.
Soc. Chimie Biol. 16: 1553-1557. Bot. 42: 869-873.
BONNER, D. M. 1938. Relation of environment and of the DUHAMET, L. 1955. Variations des besoins nutritifsdes
physical propertiesof syntheticgrowth substances to tissus vegetaux en fonctionde la taille des explantats.
the growthreaction. Bot. Gaz. 100: 200-214. Annee Biol. 31: 123-143.
BOURIQUET, R. 1954. Action de diverses substances syn- FAIRBROTHER, F., AND H. MASTIN. 1923. The swelling of
thetiques sur le development des tissus vegetaux agar-agar. Jour. Chem. Soc. 132: 1412-1424.
cultivesin vitro. Ann6e Biol. 30: 371-383. FROST,D. V., AND F. C. MCINTIRE. 1944. The hydrolysis
BURKHOLDER, P. R., AND L. G. NICKELL. 1949. Atypical
of pantothenate: a first order reaction. Relation to
growth of plants. I. Cultivation of virus tumors of thiamin stability. Jour. Amer. Chem. Soc. 66: 425-
Rumex on nutrientagar. Bot. Gaz. 110: 426-437.
427.
CAPLIN, S. M. 1947. Growth and morphologyof tobacco
GAUTHERET, R. J. 1941. Sur le repiquage des cultures des
tissue culturesin vitro. Bot Gaz. 108: 379-393.
tissus d'Endive, de Salsifis et de Topinambour. C. R.
, AND F. C. STEWARD. 1948. Effectof coconut milk
Aced. Sci. Paris. 213: 317-318.
on the growthof explants from carrot root. Science
108: 655-657. . 1942a. Hetero-auxineset cultures de tissus ve'ge
- AND . 1949. A technique for the controlled taux. Bull. Soc. Chimie Biol. 24: 13-47.
growthof excised plant tissue in liquid media under 1942b. Manuel technique de culture des tissus
aseptic conditions.Nature 163: 920-924. vegetaux. 171 pp. Masson et Cie. ed. Paris.
- AND . 1952. Investigationson the growth . 1944. Recherches sur la polarite des tissus vege-
and metabolismof plant cells. II. Variables affecting taux. Rev. Cytol. et Cytophys.ve'get.7: 45-217.
the growthof tissue explants and the developmentof . 1953. Recherches anatomiques sur la culture des
a quantitative method using carrot root.. Ann. Bot. tissus de rhizomes de Topinambour et d'hybrides de
N.S. 16: 219-234. Soleil et de Topinambour. Rev. Gen. Bot. 60: 129-173,
COOIL, B. J. 1952. Relationships of certain nutrients, 193-238.
December,1956] NITSCH AND NITSCH-GROWTH OF EXCISED TISSUES 851