INTERACTION BETWEEN HELIANGINE AND PYRIMIDINES IN ADVENTITIOUS ROOT FORMATION OF PHASEOLUS CUTTINGS

1. Heliangine at 110^–4 M promoted the adventitious rootformation in hypocotyls of cuttings taken from light-grown (1,900lux) Phaseolus mungo seedlings. The promotion was almost completelyreversed by 310^–4 M uracil, uridine, cytidine, oroticacid or 610^–4 M carbamoyl DL-aspartic acid, and partlyby 310^–4 M thymine or thymidine. Neither 310^–4M cytosine, adenine, adenosine, guanine, guanosine nor a combinationof 310^–4 M carbamoyl phosphate and 310^–4 M L-asparticacid reduced the promotion by heliangine.
2. Uracil did not reducethe inhibiting effect of heliangine onthe indoleacetic acidinduced elongation of etiolated Avenacoleoptile sections.
3. Helianginein an aqueous uracil solution was recovered unchangedafter24-hr incubation at room temperature.
4. The root formation ofPhaseolus cuttings was promoted also by2-thiouracil and 5-fluorouracil.The effect was reversed byorotic acid or carbamoyl asparticacid, but not by carbamoylphosphate plus aspartic acid.
5. Ribonucleaseat 100 µg/ml increased the number of rootsprotruded fromhypocotyls of cuttings by about 260%.
6. A possible interpretationfor the promotion of root formationby heliangine is offered.

¹ Contribution No. 15 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. ² Dedicated to Prof. Dr. H. SODING in commemoration of the 70thbirthday.     

PROMOTION OF ADVENTITIOUS ROOT FORMATION BY HELIANGINE AND ITS REMOVAL BY CYSTEINE

1. Heliangine at 10^–4M promoted the adventitious root formationin hypocotyls of cuttings taken from light-grown (1,900 lux)seedlings of Phaseolus mungo. The promotion was almost completelyreduced by simultaneously supplied 310^–4M cysteine or1.510^–4M cystine, but not suppressed by 310^–4Mof reduced glutathione, alanine or serine.
2. A 4 hr pretreatmentwith 310^–4M cysteine made Phaseoluscuttings less sensitiveto heliangine, but cysteine suppliedafter the treatment withheliangine brought about no effecton the action of heliangine.
3. Cysteine also removed the inhibiting effect of heliangineonthe indoleacetic acid-induced elongation of etiolated Avenacoleoptile sections.
4. In an aqueous solution heliangine formedan addition productwith cysteine, indicating that cysteinecan inactivate helianginewithout any biological processes.
5. On Phaseolus adventitious rooting, no effect was observedofp-chloromercuribenzoic acid, N-ethylmaleimide, 1,4-naphthoquinone,coumarin or penicillin. Reactivity toward sulfhydryl groupsalone does not qualify a substance to be a promotor of rootformation.
6. Maleic hydrazide at 10^–4M promoted root formation,butits effect was not removed by cysteine.

¹ Contribution No. 13 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Koishikawa, Tokyo.     

The Effect of Prolonged Chilling on Water Movement and Radial Growth in Trees

1. The woody shoots of young saplings of Fraxinus.excelsior andAcer Pseudo-platanus in pots were subjected to continuous coolingto about 2° C. during the growth season, with the resultthat radial growth was almost completely inhibited throughoutthe woody stem.
2. The chilling did not adversely affect extensiongrowth exceptthat it was later in commencement and proceededmore slowly.
3. If the temperature around the stem is loweredfrom 2° C.to 0° C., water conduction is cut down tosuch an extentas to cause wilting of the leafy shoots; turgidityis recoveredwhen the temperature is again raised to 2°C.
4. This wilting effect is discussed particularly in relationtothe part played by living cells in the upward movement ofwaterin the wood.

     

Biological activity of phyllosinol, a phytotoxic compound isolated from a culture filtrate of Phyllosticta sp.

1. Phyllosinol is a phytotoxic metabolite of Phyllosticta sp. Thissubstance at 100 µg/ml produced dark grey necrotic lesionson the leaf of red clover. Sensitivities of various plant speciesto phyllosinol differed both quantitatively and qualitatively.
2. Phyllosinol reduced root growth in rice seedlings by 60% at10^–4 M, whereas stimulation of root elongation occurredat a concentration range from 10^–9 to 10^–5 M.
3. Phyllosinolat 2.5x10^–4M promoted adventitious root formationin epicotylsof Azukia cuttings by about 100%. Promotion waspartly reducedby simultaneous application of cysteine.
4. IAA-induced elongationof isolated Avena coleoptile sectionswas inhibited by phyllosinolat a concentration range from 10^–5to 10^–3M.
5. Sulfhydrylcompounds, i.e. cysteine and glutathione relievedinhibitioncaused by phyllosinol in IAA-induced elongation ofAvena coleoptilesections.
6. GA₃-induced elongation of wheat leaf sections wasslightly inhibitedby phyllosinol at 10^–4M.
7. Phyllosinolalso has antibiotic activity. Among the organismstested, Phycomycetesand Gram-negative bacteria appeared mostsusceptible to phyllosinol.

(Received April 21, 1970; )     

Absorption and Assimilation of Inorganic Nitrogen from Different Sources by Storage Root-Tissue

1. The rate of ammonium and nitrate absorption and assimilationby storage tissues of sweet potato tubers, radish, and carrotroots cultured in aerated dilute solutions (5 mg. eq./1.) ofNH₄Cl, KNO₃ or NH₄NO₂ at 25° C. and the effect of the sourceof nitrogen on the nitrogenous composition of the tissues werestudied.
2. Both ions were absorbed at more or less equal ratesunder theprevailing experimental conditions by cells of thedifferentplant materials used.
3. The rate of nitrate assimilationwas lower than absorption,leading to the accumulation of nitrate-Nin the cells of alltissues examined. Ammonium-N, on the otherhand, was assimilatedalmost as soon as it entered the cellsof sweet potato tubersand carrot roots, and there was onlya small increase in thelevel of arnmonium-N; but in radishroot cells ammonium assimilationlagged very much behind absorption,resulting in a great increasein the level of arnmonium-N.
4. Sweetpotato tubers and carrot tissues were shown to be adaptedtothe utilization of more ammonium than nitrate nitrogen, butradish root tissues are adapted to the utilization of more nitratenitrogen.
5. Ammonium or nitrate-N caused similar increases inthe complexorganic-N level (protein-+rest-N) in radish root,but the rateof synthesis was higher with ammonium in sweetpotato tubersand with nitrate in carrot roots.

Furthermore, with both sources changes in the content of amideand amino-N were negligible in radish tissues of both series.In contrast to radish, carrot tissues showed marked increasesin the two fractions. The increase of these fractions in sweetpotato tuber tissues was much lower. The marked contrast in nitrogenous composition in the threedifferent plant materials fed with ammonium and nitrate saltsseparately indicated that the actual course of protein metabolismis most probably dependent on the source of nitrogen utilized.     

The Time Factor in Studies of Growth Inhibition in Excised Organ Sections

1. Apprehension over the adequncy of current techniques stimulateda detailed study of the time factor in the arsenate inhibitionof growth and respiration in excised stem and root sectionsof Pisum sativum.
2. Growth inhibition by arsenate sets in veryslowly, its rateof onset being related to the molar concentration(C) of arsenateate by the relation where T₅₀ is the time taken in hours to reduce the growthrateto 50 per cent of the control and K is a constant. An explanationof the physiological basis of this relationship is attempted.
3. Estimates were made of the final steady growth rate (relativeto control) in various arsenate concentrations. The inhibitionscalculated from this rate are held to approximate to the truearsenate effect and are shown to be very different from thosecalculated from ‘total growth’ measures.
4. Respirationof growing stem sections is not inhibited by thelow arsenateconcentrations that inhibit growth. Some inhibitionis indicatedat high concentrations (3 ? 10^–4M. and over)but onlyafter 15-20 hours of exposure.
5. Two per cent sucrose has noeffect on the arsenate inhibiitionof stem growth. Sucrose,however, markedly stimulates respirationin stem sections, butthis stimulation is prevented by arsenate.
6. The misinterpretationswhich may arise as a result of ignoringthe time factor in inhibitionstudies in excised organ sectionsare discussed and the desirabilityof constructing completegrowth curves in all such studies isstressed.

     

STUDIES ON COMPONENTS OF RESPIRATORY SYSTEM OF BEGONIA LEAVES

Investigations were made of the properties of diaphorase, cytochromec reductases, cytochrome c oxidase, and other components ofelectron transfer system in various fractions of leaf homogenateof Begonia semperflorens.

1. All the fractions tested showed the existence of cytochromec oxidase, succinic- and reduced diphosphopyridine nucleotide-cytochromec reductases, and diaphorase. Activities of these enzymes werefound to be associated mainly with the particulate fractions.The particulate fractions showed, in particular, a capacityof reducing oxidized cytochrome c with fumarate, malate, -ketoglutarate,ß-hydroxy-butyrate, and citrate.
2. Optimum pH foroxidation of cytochrome c by the particulatefractions was foundto be 5.5, while that for reduction was7.2.
3. The activityof cytochrome c reductase was partially suppressedby malonate.Partial inhibition of cytochrome c oxidase wascaused by azideand cyanide, the inhibitory effects observedbeing strongerwith particulate fractions than with solublefractions.

(Received August 11, 1962; )     

Factors Controlling Flowering of the Chrysanthemum: I. THE EFFECTS OF PHOTOPERIOD AND TEMPORARY CHILLING

1. Experiments are described which indicate that a temporary exposureto low temperature (vernalization) hastens inflorescence budinitiation in the Chrysanthemum, as measured by the time tothe macroscopic appearance of the bud and also by the numberof leaves produced. This effect is found in both long-and short-dayconditions.
2. In the absence of vernalization plants kept inshort day assumea diageotropic growth habit and remain vegetativefor long periods,frequently for much more than one year. Unvernalizedlong-dayplants also remain vegetative but have a normal geotropicreaction.
3. While the day-length effect is less important forinflorescencebud initiation, the opening and further developmentof budsformed in long day depend normally on subsequent day-lengthtreatment.
4. A vernalization period of only three weeks appearsto be fullyadequate.
5. The low-temperature treatment may begiven discontinuously,and evidence to hand appears to indicatethat it is more effectiveif given during the dark phase thanduring the light phase.Hence de-vernalization by temperaturesof about 20–25°C. does not appear to take place.
6. There is evidence that little or none of the stimulus is carriedover from one year to another.
7. The results are discussed inrelation to the auxin metabolismof the plant and also withregard to the absence in the literatureof previous mentionof the cold requirement.

The author is indebted to Professor F. G. Gregory and to Mr.F. J. Richards for their stimulating interest and helpful suggestionsin the course of this work, and to Dr. M. Holdsworth for providinghim with the results of earlier unpublished work. Messrs. H. Woolman Ltd. kindly supplied some of the plant materialused.     

STUDIES ON THE MECHANISM OF GROWTH INHIBITING EFFECT OF LIGHT

1. A substance which inhibits indoleacetic acid (IAA)-and naphthaleneaceticacid (NAA)-induced elongation of Avena coleoptile section andIAA-induced Avena coleoptile curvature was found in an ethersoluble neutral fraction of water extract of sunflower leavesand in agar blocks containing the diffusate from young sunflowerleaves.
2. This substance also inhibits the growth of isolatedsunflowerepicotyl.
3. The R_f value (0.9) of the substance ona paper chromatogramdeveloped with ammoniacal iso-propanolindicates that it isidentical with the inhibitor reported byAUDUS et al. (1956),but not with inhibitor-ß.
4. Theinhibitor can be transported from leaf to stem, and thetransportseems to be accelerated by illuminating the leaf.
5. The auxindiffused from sunflower leaf into agar block may beidenticalwith IAA.
6. A substance, which has the same properties as theinhibitorfrom sunflower leaf, was obtained in crystalline formfrom theleaf of Jerusalem artichoke.
7. The mechanism of growthinhibition caused by this crystallinesubstance seems to involveinactivation of a sulfhydryl group.
8. The reason why the stemgrowth of sunflower seedlings is reducedby strong light isdiscussed: the amount of the inhibitor transportedfrom leafto stem is increased under strong light, and in thestem, growthinhibition is caused by a direct effect of thisinhibitor ongrowth and by its inhibiting effect on the transportof IAAfrom leaf to stem.

¹ Present address: Botanical Garden, Faculty of Science, Universityof Tokyo, Tokyo (Received February 15, 1961; )     

Effect of Chilling on DNA Endoreplication in Root Cortex Cells and Root Hairs of Soybean Seedlings

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C – 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C – 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C – 8C DNA nuclei predominated while in chilled plants an additional population of 2C – 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred. This revised version was published online in July 2006 with corrections to the Cover Date.     

Studies on Plant-Growth Hormones: VI. THE NATURE OF INHIBITOR-{beta} IN POTATO

1. The chemical nature of a plant growth inhibitor in potato tuberpeel, the ‘Inhibitor-ß’ which commonlyoccurs on chromatograms of many plant extracts, has been examined.
2. The inhibition, as indicated by bioassays using wheat coleoptilesections, could not be associated with any particular compound,but was partly or entirely due to a complex mixture of aliphaticacids.
3. . Azelaic acid and the coumarin, acopoletin, were isolatedtogetherwith a new substance, Acid A; degradative evidenceis not sufficientto enable a complete structure to be proposedfor this acid,but it appears to be an unsaturated polyhydroxyfatty acid.
4. The growth of coleoptile sections in solutionsof ßat several concentrations was examined over thefirst 7 hoursof growth. Inhibition did not occur until 4 hours;visible damageto the cells of the tissues appeared after thisperiod. Whenß was examined in a mixture with 3-indolylaceticacid,inhibition was evident after 1 hour. These results areinterpretedand the chemical system in which ß mayoperate ingrowth is briefly considered.

     

"GIGANTISM" OF CHLORELLA VULGARIS I. MECHANISM OF INDUCTION OF CIGANTISM

1. Based on the microscopic observations, two stages, "giant cellstage" and the subsequent "palmelloid body stage", were distinguishedin the process of formation of giant Chlorella induced by theaddition of sugars. The "giant cell" is much larger in sizethan the control cell, but the other morphological featuresare the same as those of the latter. The "palmelloid body" isa form composed of many conjoined autospores.
2. When a highconcentration of glucose was maintained in the medium,gigantismwas also maintained. Under this condition, the algashows acyclic transformation between "giant cell" and "palmelloidbody"without returning to the small single cells.
3. Large amountsof carbohydrate composed of hexose were foundto be accumulatedin the giant algal cells, and it was inferredthat this carbohydrateaccumulation causes greater enlargementof cell volume as comparedwith control cells.
4. Uronic acids, which were found to be absentin the control cells,were formed and lost in the cells culturedin the glucose mediumin parallel with the appearance and disappearanceof gigantism.
5. Pectic substances, from which uronic acids areconsidered tobe derived during the extraction procedure, werefound to bepresent only in giant Chlorella.
6. The conjoinedautospores in giant Chlorella (at the palmelloidbody stage)were separated to some extent by the addition ofEDTA, and theresulting cells were similar to control Chlorellacells.
7. Basedon these results it was inferred that inductive formationofthe pectic substances is causally related with the appearanceof "palmelloid body".

¹ Present address: Department of Chemistry, College of GeneralEducation, Osaka University, Toyonaka, Osaka.     

The Growth Substances separated from Plant Extracts by Chromatography: II. THE COLEOPTILE AND ROOT ELONGATION PROPERTIES OF THE GROWTH SUBSTANCES IN PLANT EXTRACTS

1. 1. A method for the running of ‘strip’ chromatogramsof plant extracts, as large-scale sources of the naturally occurringgrowth substances accelerator () and inhibitor ß(ß), and the elution of these substances togetherwith indole-3-acetic acid (IAA), is described. A method is givenfor the testing of the pea root section extension propertiesof these growth substances.
2. 2. Coleoptile and root sectionextension tests over a completeconcentration range are donefor , ß, and eluted IAA,and mixtures of and ßwith IAA or indole-3-acetonitrile(IAN) are tested for coleoptilesection extension.
3. 3. promotes at low concentrations andinhibits at high concentrationsboth coleoptile and root sectionextension and the coleoptilesection extension induced by IAAor IAN. ß inhibitscoleoptile and root section extensionover the whole concentrationrange; it also inhibits IAA andIAN induced coleoptile sectionextension.
4. 4. The extensionof coleoptile sections in mixtures of or ßwith IAAis measured at a number of time intervals. , aloneand withIAA, has its greatest promoting effect in the earlystages andits greatest inhibiting effect in the later stagesof sectiongrowth. ß, alone, promotes the early stagesand inhibitsthe later stages of section growth and, with IAA,has its greatestinhibitory effects in the later stages.

     

CHANGES IN RESPONSE TO PURINE AND PYRIMIDINE DERIVATIVES OF CARROT ROOT CALLUS DURING SUCCESSIVE CULTURING

1. The growth of the carrot root callus which had been subculturedfor a long period (CCL) was promoted by the addition of 5l0^–8and 5l0^–7 M kinetin, whereas in the callus subculturedfor a short period (CCS) no growth promotion was observed atany concentrations of kinetin tested.
2. CCL showed an increasedgrowth in response to the applicationof kinetin, guanine, adenine,hypoxanthine, uracil, thymine,and cytosine in the presenceof fractions A and C of carrotroot extract, whereas no suchresponse was observed in CCS.CCL required fraction C to respondto uracil and probably purineand pyrimidine derivatives ingeneral.
3. The growth of CCL was promoted by kinetin, guanine,adenine,or hypoxanthine in the medium containing inositol andaminoacids mixture. In this case the growth-promoting actionof guanine,adenine, or hypoxanthine was nullified by kinetin.

(Received December 24, 1964; )     

The Effects of Sugar and Potassium on Extension Growth in the Root

1. A new technique for studying extension growth in the root isdescribed which is based on excising a zone which extends 1·5–3·0mm. from the tip. Large numbers of these segments are culturedwith different nutrient fluids in the dark at 25° C. withcontinual shaking.
2. The effects of a large number of nutrientson the growth ofthe segments have been studied, but only two,sugar and potassiumions, have been found to have stimulatingeffects.
3. The effects of water, three concentrations of sugar,and oneof potassium in air, and with an atmosphere containing5 percent. oxygen have been studied in detail in connexionwith lengthincrease, sugar absorption, content of free sugar,cellulosecontent, dry weight, and respiration.
4. It has beenshown that with increasing concentration of sugarin the medium,the rate of growth, the time during which growthproceeds, theinternal concentration, respiration, dry weight,and celluloseformation all increase. Also that potassium stimulatesthe rateof growth and respiration, and that with per cent,oxygen allthe aspects studied are depressed.
5. It is suggested that thestimulation due to sugar may be attributedto an accelerationof water absorption with a complementaryincrease in celluloseformation. It is further suggested thatsugar accelerates waterabsorption by accumulating in the vacuoleand thus sustainingthe osmotic pressure of the vacuolar sap.It is further suggestedthat potassium stimulates growth byincreasing water absorptionthrough an effect on respiration.The effect of respirationin this connexion may be to promotethe transport of water directlyto enhance the osmotic pressureof the sap by inducing an accumulationof inorganic ions inthe vacuole.

     

Correlation, between {beta}-galactosidase and auxin-induced elongation growth in etiolated pea stems

ß-Galactosidase, -galactosidase, ß-glucosidaseand ß-xylosidase were studied in relation to auxin-inducedelongation of etiolated pea stems. Frozen-thawed sections wereincubated in substrate solutions and individual enzyme activitywas determined. Results were as follows:

1. Only ß-galactosidase activity was remarkably enhancedby 2,4-D in the concentration range which induced remarkableelongation.
2. Auxin-induced increase in ß-galactosidaseactivityreached maximum after 1 hr treatment of the sectionwith 2,4-D(10^–5 M), and was nearly constant afterwards.
3. Auxin-enhanced ß-galactosidase activity was observedeven if auxin-induced apparent elongation was osmotically suppressedby mannitol.
4. Correlation analysis indicated that ß-galactosidaseactivity had a high positive correlation with auxin-inducedelongation and increase in lateral face area of the section,while none of the other enzyme activities did.
5. Measurementof in vivo distribution of four glycosidase activitiesin intactthird internode of the epicotyl made clear that onlyß-galactosidaseshowed maximum activity in the zonewhere endogenous elongationwas maximum.

(Received February 5, 1976; )     

The Organic Acid Metabolism of Cox's Orange Pippin Apples: I. SOME EFFECTS OF THE ADDITION OF ORGANIC ACIDS TO THE PEEL OF THE FRUIT

1. The basic respirations (CO₂-output and O₂-uptake) of Cox'sOrangePippin apples and of the peel tissue prepared from themwerecompared in fruit in various stages of development, bothinitiallyand after storage at 12°C. Both show the samegeneral trend,although as the apples become mature the peakvalue of the respirationclimacteric tends to rise in the wholefruit and fall in thepeel.
2. The effect of adding malate orcitrate on the respiration ofthe same samples of peel was studied.
3. Three broad stages of development were observed. During thefirst stage (petal fall to 60 days after) the metabolic patternappears to be different from the two later stages. Here O₂-uptakeas well as C₂-output are influenced by the addition of bothmalate and, to a considerably less extent, citrate. In stage2 (60–125 days from petal fall), the malate effect (CO₂-output)is small until after detachment from the tree, when it risessharply. In stage 3 (125 days to full maturity) the malate effectfollows the course expected for earlier work, namely, it developsat the same time as the climacteric rise in respiration. Thepossible reasons for the different behaviour of the peel atthe three stages is discussed.
4. Results were similar in generaltrend for Cox's Orange Pippinapples grown on different rootstocksand under different culturalconditions.
5. It is suggested thatthe malate effect is most active in theepidermal and hypodermaltissues of the fruit.

     

The Water Permeability of Unplasmolysed Tissues2

1. The permeability of unplasmolysed cells of beetroot, v. ‘CrimsonGlobe’, was determined from the rate of water loss ofbeet slices on placing in sucrose solutions having O.P. greaterthan the suction pressure of the beet. The absolute values obtainedwere about 0?7µ³ water per µ² cell-surface per hourper atmosphere osmotic pressure difference, i.e. 0?7 µ/hr./atm.
2. The permeability of similar beet cells plasmolysed withintheircell walls was found to be about 13µ/hr./atm.
3. Thepermeability of beet cells which had been plasmolysed andallowedto recover was shown to be approximately the same asthat ofunplasmolysed cells.
4. The hypothesis is advanced that the increasein water permeabilityon plas-molysis is due to those partsof the plasma-membranewhich had formerly been pressed againstthe micelles of thecell wall becoming free and able to takepart in water transfer.
5. The energy requirement for the maintenanceof an excess hydrostaticpressure of five atmospheres withina cell by its vital activitywas shown to be about one-tenthof the total respiratory energyreleased in freshly cut beetslices.

     

Microbiological Synthesis of Fat: THE EFFECTS OF NITROGEN LEVEL ON THE METABOLISM OF PENICILLIUM LILACINUM THOM IN A SUCROSE MEDIUM

1. A study has been made of the relationships between the synthesesof carbohydrate, protein, and fat by Penicillium lilacinum Thomin presence of different amounts of sodium nitrate us a definedsucrose salts medium.
2. Under the defined experimental conditionsincreases in the concentrationof NO^–₂ in the medium werefollowed by increases in therates at which nitrogen and sugarwere taken up by the fungus,in the quantities assimilated,and in total and protein nitrogenin the felt. These conditionsprevailed so long as unassimilatedsugar was available.
3. Mediaof lower NO^–₃ concentration (for example, 0·32or 0·64 per cent. (w/v) NaNO^–₂;) yielded feltsricher in carbohydrate than were those grown in media of higherNO^–₂; content (0·96 or 1·28 per cent. (w/v)NaNO₃ The carbohydrate content of the felts increased graduallyuntil the sugar in the medium was exhausted; carbohydrate contentthen decreased.
4. Media of lower NO^–₃; concentration weremore conduciveto fat synthesis than those of higher NO^–₃;content.

     

CHANGES IN THE RESPONSE OF CARROT ROOT CALLUSES TO THE ROOT EXTRACT OCCURRING DURING THEIR SUCCESSIVE CULTURES

1. Alcohol extract of carrot root promoted the growth of the carrotroot callus which had been succesively cultured for more than18 months (CCL) on the medium containing WHITE'S inorganic salts,sucrose, yeast extract and 2, 4-D, but only a weak promotionwas observed for the growth of the carrot root callus whichhad been cultured for less than 14 months (CCS).
2. The activesubstances were fractionated by Amberlite IR-120and AmberliteIRA-400 into four fractions; C, D, E, and F. Eachfraction seemedto act synergistically to produce the effectof the whole carrotroot extract on the growth of CCL.
3. Fraction F of the carrotroot extract, which was adsorbed byAmberlite IRA-400 but notby Amberlite IR-120, promoted thegrowth of CCL in the presenceof other fractions, but had noeffect on the growth of CCS.So the different responses to thealcohol extract of the carrotroot calluses having differentlengths of successive cultureperiod seemed to depend mainlyon the ability of respondingto fraction F.
4. Using four strains of carrot root callusesof different origin,it was ascertained that different responsesof carrot root callusesto fraction F depended on the lengthof their culture and noton their strain-specific characters.
5. The substances active for the growth of CCL in the carrotrootextract passed through a dialysis membrane. These substanceswere little affected by autoclaving and remained in the aqueouslayer when shaken with several organic solvents: n-butanol,ethyl acetate, chloroform, benzene, ethyl ether and carbon tetrachloride.
6. Alcohol extract of carrot root also promoted the growth ofcarrotroot explant, tobacco stem callus and sunflower crowngall tissue.

(Received December 24, 1964; )