Growth and Cellular Differentiation in the Wheat Coleoptile (Triticum ...

Growth and Cellular Differentiation in theWheat Coleoptile (Triticum vulgare)

II. FACTORS INFLUENCING THE GROWTH RESPONSETO GIBBERELLIC ACID, KINETIN AND

INDOLYL-3-ACETIC ACID

S. T. C. WRIGHT

Agricultural Research Council, Plant Growth Substance and Systemic Fungicide Unit, WyeCollege, University of London, Nr. Ashford, Kent

WITH THREE FIGURES IN THE TEXT

Received 5 May 1965

ABSTRACT

The sequential growth response of excised wheat coleoptiles during growthand cellular differentiation to solutions of gibberellic acid, kinetin, and indolyl-3-acetic acid is further analysed in this paper.

Excised young coleoptiles which possess a low potential growth-rate (controls)respond to gibberellic acid and kinetin, whereas excised older coleoptiles whichhave a high potential growth-rate respond to indolyl-3-acetic acid. The dif-ference between these results and those reported for pea-stem sections is dis-cussed.

To obtain a well-defined separation of the sequential growth response togibberellic acid and kinetin it is necessary to grow the wheat coleoptiles at22-5° C. At a higher temperature (250 C) the two optimal responses tend tooverlap. It is suggested that this difference is due to a delayed onset of celldivision at the lower of the two temperatures.

Substances diffusing from the primary leaves contained within the excisedcoleoptile do not interfere with the sequential growth response of the coleoptile.Differential uptake of the growth regulators into the older coleoptiles does notexplain the lower growth response stimulated in these coleoptiles by gibberellicacid and kinetin as compared to the high activity of indolyl-3-acetic acid.

The possible importance of gibberellic acid in primary cell-wall growth isdiscussed.

INTRODUCTION

EARLIER publications (Wright, 1961 a, b) showed that the growth of theintact wheat coleoptile can be divided into three phases: (i) an early periodof cell enlargement (approx. 0-30 hours after sowing), (ii) a period of cellenlargement accompanied by cell division (approx. 30-60 hours after sow-ing), and (iii) a final period of cell enlargement (approx. 60-120 hours aftersowing). Details were also given of how the wheat coleoptile respondssequentially to gibberellic acid, kinetin, and indolyl-3-acetic acid (IAA)

Journal of Experimental Botany, Vol. 17 No. 50, pp. 165-76, February 1966.

Downloaded from https://academic.oup.com/jxb/article-abstract/17/1/165/498495by gueston 02 April 2018

166 Wright—Growth and Cellular Differentiation

during its growth and cellular differentiation (Wright, 1961a). The greatestsensitivity to gibberellic acid was found to be correlated with the period ofcell enlargement which precedes cell division, whereas kinetin elicited thegreatest response from coleoptiles in which many of the cells were about todivide or were dividing. IAA seemed to be important in the final cell-enlargement phase. In this paper it is proposed to enlarge upon the practicaldetails of these experiments and to give the results of experiments which werecarried out to test the validity of the sequential growth response.

Since the earlier work was published, van Overbeek (1962) has postulatedthat these three classes of growth regulators may be concerned in a sequentialmanner in the growth of fruit.

MATERIALS AND METHODS

Coleoptile culture method. Wheat grains (var. Eclipse) were soaked for twohours in distilled water and then sown, with their embryos oriented upper-most, on moist filter paper in 9-cm Petri-dishes. The Petri-dishes (withouttheir lids) were transferred to a box (25 X 21 X 9 in.) with a humid atmosphere(90 per cent relative humidity) and maintained at 22-5° C in darkness untilrequired.

Method of testing response to growth-regulating substances. Whole coleoptileswere excised using the epiblast as a guide (Wright, 19616) and the coleoptilesplaced on moist filter paper until sufficient numbers had been cut for theexperiment. Coleoptiles were measured under a binocular microscope witha micrometer eyepiece or with a millimetre rule to the nearest 0-5 mm,depending on age. Five coleoptiles were placed on a 2 X 1 in. strip of WhatmanNo. 1 filter paper and inserted in a 3 X J in. test-tube containing o-6 ml of thetest solution. (The purpose of the filter-paper strip was to prevent the smallcoleoptiles from drying out on the sides of the tube.) Five, occasionally four,replicates of five coleoptiles were used per treatment. The tubes were sealedwith 'Parafilm' in which a small hole was pierced near the centre to allowventilation. The tubes were rotated at 1 r.p.m. on a klinostat in dim red light(emission > 645 m/x) at 260 C for 24 hours. From the mean length of the fivecoleoptiles before and after treatment the percentage increase in mean lengthof the coleoptiles was calculated for each replicate (i.e. percentage increaseover original length).

Method of preparing growth-substance solutions. An aqueous solution ofgibberellic acid (io~4 M = 3-46 mg/100 ml) was stored at 2° C for up tofour weeks. Kinetin crystals do not dissolve at room temperature and it wasnecessary to heat the solution in a water bath at 95° C for five minutes. Freshsolutions were prepared for each test since the solutions crystallized out inthe refrigerator (3-3 x io"4 M = 7-16 mg/100 ml). IAA was kept as a io~3 Maqueous solution of the sodium salt at 1° C for up to four weeks (17-5 mgIAA crystals dissolved in distilled water with 1 ml M/10 NaOH and madeup to 100 ml).


in the Wheat Coleoptile (Triticum vulgare). II

RESULTS

I 6 7

(i) Analysis of the sequential growth response data. In earlier experiments(Wright, 1961a) dosage-response curves for gibberellic acid, kinetin, andIAA were determined for a series of coleoptile ages for each growth regulatorfrom 18 to 78 hours after sowing. Fig. \a and \b show dosage-response

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2 S.E. limits.O = gibberellic acid; A = kinetin)

curves for gibberellic acid and kinetin of coleoptiles excised 18 hours and36 hours after sowing (dosage-response curves for IAA are not shown) andby referring to such curves it was possible to determine the concentrationsgiving the optimal growth response over a range of coleoptile ages for eachgrowth regulator. It was found that there was little change in the optimalconcentration of each of the three growth regulators with increasing age ofthe coleoptile. The optimal concentrations were io~5 M for gibberellic acid,io~4 M for kinetin and io~4 M for IAA. By using only the optimal concentra-tion for each growth regulator it was possible to determine the growthresponse of the coleoptile at 18, 30, 42, 54, 66, and 78 hours after sowing inone large-scale experiment and thereby eliminate any inter-experimentalvariation in coleoptile sensitivity (Wright, 1961a). The results which showthe sequential growth response of the excised wheat coleoptile to gibberellicacid, kinetin, and finally IAA are reproduced again in Fig. 2b, with thegrowth response expressed as percentage increment over control.1 When theresults are plotted as percentage increase over original length they appear asin Fig. 2a. This figure shows clearly that the large growth responses to gib-berellic acid and kinetin which occur in young coleoptiles (Fig. zb) are partly

1 Percentage increment over control = percentage increase over original length of treatedcoleoptiles—percentage increase over original length of control coleoptiles.



a reflection of the low potential growth-rate of the excised coleoptile at thisstage as illustrated by the growth of the water controls (Fig. 2a).

(ii) The reproducibility of the sequential growth response to gibberellic acid,kinetin, and IAA. The sequential growth response illustrated in Fig. zb hasbeen repeated ten times in this laboratory, using wheat (var. Eclipse) fromthe harvests of several different years. The results have always shown that

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FIG. 2. The response of the wheat coleoptile to exogenous solutions of gibberellic acid,kinetin, and indolyl-3-acetic acid: (a) with growth responses expressed as percentage increaseover original length; and (6) growth responses expressed as percentage increment overcontrol length, to illustrate the sequential growth response. The vertical lines indicate the

2 S.E. limits.0 = water controls; O = gibberellic acid, io~5 M; A = kinetin, io~4 M;

• = indolyl-3-acetic acid, io~4 M)

a response to gibberellic acid and kinetin precedes a response to IAA. How-ever, the time elapsing between the gibberellic acid and kinetin peaks hasbeen found to vary and on some occasions the two peaks have almost coin-cided. The reason for this variation in the time of these peak responses hasbeen partially elucidated.

Two treatments which had no effect on the position in time of the gib-berellic acid and kinetin peaks were (i) exposure to red light as compared


in the Wheat Coleoptile (Triticum vulgare). II 169

with darkness during the period of treatment in the klinostat, (ii) ventilationof the test-tubes containing the coleoptiles by piercing a hole in the centre ofthe 'Parafilm' cover, compared with no ventilation in the klinostat. It mustbe noted that there was a need for ventilation in tubes containing the largercoleoptiles (i.e. 60 hours and above) to obtain a maximum response to IAA.

The third treatment was a comparison between two growing temperatures.Normally, all coleoptiles were grown at 22-5° C (see Methods) and these

24 36 48 24Age of coleoptiles (h)

36

FIG. 3. The reproducibility of the gibberellic acid and kinetin sequence (a), (c), and (d)with coleoptiles grown at 225° C; (b) with the coleoptiles grown at 25° C.

O = gibberellic acid, io~5 M; A = kinetin, io~4 M)

when excised gave a peak response to kinetin somewhere between 36 and 42hours after sowing (Fig. 3a). When the coleoptiles were grown at 25° C, how-ever, the kinetin peak response was found to occur between 24 and 30 hoursafter sowing and almost overlapped the gibberellic acid peak (Fig. 36). (Inthe experiment represented in Fig. 26 the coleoptiles were grown at 230 Cand the peak response to kinetin was at 30 hours.) Thus the growing tempera-ture was found to be very critical and a difference of 2-5° C produced distinctlydifferent results. Fig. 3c and 3^ represent repeat experiments of the experi-ment in Fig. 3a, and as can be seen there was generally a falling off in thegrowth response stimulated by gibberellic acid with increasing coleoptile age(except in Fig. ^d where there was a peak response at twenty-four hoursafter sowing). Kinetin gave a peak response at approximately 36 hours aftersowing in all three experiments.

(iii) Effect of substances diffusing from the primary leaves enclosed withinthe coleoptile on the growth response of the coleoptile to gibberellic acid, kinetin,



and IAA. A possible criticism of the experiments which demonstrated thesequential growth response concerns the fact that the primary leaves wereretained within the coleoptiles during the period of growth-substance treat-ment. For technical reasons the leaves were retained since it was found to beimpossible, except with very young coleoptiles (i.e. 18 hours after sowing),to remove the leaves without damaging the coleoptiles. Now it is conceivablethat during the early growth of the primary leaves within the coleoptilea substance is produced at a certain growth stage which could diffuse intothe medium when excised and react synergistically or antagonistically witha growth regulator in coleoptile growth. For instance if a synergist to kinetinwas produced by the leaves at 36 hours after sowing it would lead to theobserved optimal growth response of the excised coleoptile to kinetin at thisstage. The following experiments were carried out to determine whether suchsubstances diffuse from the primary leaves and influence the response of theexcised coleoptile to the growth regulators. These determinations were madeat the three critical stages of coleoptile growth (i.e. 18 hours after sowing)which is the stage responding most actively to gibberellic acid; 36 hoursafter sowing when the coleoptile is most sensitive to kinetin (Fig. 3a, c, and d);and 66 hours after germination when the coleoptile responds to IAA.

With the youngest coleoptiles (18 hours after sowing) four treatments weregiven, (i) the primary leaves were left undisturbed, (ii) the primary leaveswere teased out of the coleoptile but retained in the test-tube with the treat-ment solution, (iii) the primary leaves were teased out and discarded, and(iv) as (iii) except that leaves from five 36-hour coleoptiles were placed in thetest-tube. The last treatment was included to see if any substance diffusingfrom 36-hour leaves might cause the 18-hour coleoptile to respond prefer-entially to kinetin. The results shown in Table 1 indicate that the response ofthe coleoptiles to gibberellic acid, kinetin, IAA, and distilled water in treat-ments ii, iii, and iv was not significantly different from control coleoptiles(i.e. treatment i). When the leaves were teased from the coleoptiles as intreatments ii, iii, and iv the kinetin response was slightly lower than thatgiven by gibberellic acid (i.e. 56, 59, and 57 per cent as compared with 65,64, and 60 per cent); these sets of figures were significantly different at the5 per cent level whereas in treatment i although the kinetin response waslower than the gibberellic acid response there was no significant difference.

With coleoptiles excised 36 hours after sowing it was found to be im-practicable to tease out the primary leaves since the coleoptiles were too long(i.e. 3 mm). Three treatments were chosen, (i) control coleoptiles with theirenclosed primary leaves left undisturbed, (ii) coleoptiles with their enclosedprimary leaves plus an extra set of primary leaves dissected from five 36-hourcoleoptiles, (iii) coleoptiles with their enclosed primary leaves plus leavesfrom ten 18-hour coleoptiles. The growth response to gibberellic acid,kinetin, IAA, and distilled water in treatments ii and iii was not significantlydifferent from the controls (i) (Table 1). The growth response to kinetin wasalways greater than that given by gibberellic acid whatever the treatment and


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this again suggested that the retention of the primary leaves was not the causenor in any way influenced the sequential growth response.

In the larger coleoptile (i.e. 66 hours after sowing) the effect of the primaryleaves was investigated by using coleoptile sections. Three treatments wereemployed using i-cm sections cut 3 mm from the tip of the coleoptiles, (i)the leaves were left within the sections (controls), (ii) the enclosed leaveswere pushed out with the aid of fine glass rods and the leaf sections discarded,(iii) the enclosed leaves were pushed out with glass rods and the coleoptilesections left threaded on them during treatment. The removal of the primary

Treatment

Water controls

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Kinetin 10^ M

IAA 10-4 M

TABLE 2

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leaves (treatments ii and iii) resulted in a significant reduction in the growthresponse to all three growth regulators and also in the water controls. Whenthe sections were left threaded on glass rods (treatment iii) the growthresponse was greatly impaired, particularly in the water- and kinetin-treatedsections. Nevertheless, whether the primary leaves were present or absent,IAA was always the most active growth regulator at this stage.

(iv) Influence of the rate of uptake of the growth regulators by the older coleop-tiles on their response to gibberellic acid, kinetin, and IAA. To test this effectcoleoptiles excised 60 hours after sowing were cut in half and the total re-sponse of the two halves compared with the response of whole coleoptiles.The results are given in Table 2. The most pronounced difference betweenthe two treatments was in the water controls. Here the cut coleoptiles grewsignificantly more (P = o-ooi) than whole coleoptiles. Gibberellic acid andIAA gave a greater final length when the coleoptile was cut in two, whereasthe kinetin response was the same whether cut or not cut. When the resultsare expressed as percentage increment over control length the gibberellicacid, kinetin, and IAA responses are higher in the whole coleoptiles.

DISCUSSION

Purves and Hillman (1958) cut sections from Alaska pea stems at variousdistances from the apical hook and measured their growth response aftertreatment in solutions of gibberellic acid and IAA. They found sections nearthe apex responded particularly to gibberellic acid (i.e. young cells), whereas


in the Wheat Coleoptile {Triticum vulgare). II 173

sections more distant (i.e. older cells) were more sensitive to IAA. Further-more, the sections which were very responsive to gibberellic acid also possesseda high potential ('endogenous') growth-rate whereas those sensitive to IAAhad a low potential growth-rate. In the excised wheat coleoptile the patternof response to gibberellic acid and IAA with increasing cell age was similarto that of pea sections (Fig. zb), but there was a complete reversal as regardsthe correlation between potential growth-rate and growth-substance activity.Excised wheat coleoptiles with low potential growth-rates responded mostactively to gibberellic acid and those with high potential growth-ratesresponded most actively to IAA (Fig. zd). Thus it would seem that thepotential growth-rate of a tissue is not a guide to how it will respond togibberellic acid or IAA.

The low potential growth-rate of the young excised coleoptile would sug-gest that a gibberellin and/or a kinin may be severely limiting growth in theseearly stages. Later when the potential growth-rate increases (i.e. at 42 hoursafter sowing the excised coleoptile grows 60 per cent above the originallength) it begins to respond to IAA. This increase in the potential growth-ratecould indicate a build up in the levels of naturally occurring gibberellinsand kinins.

When the wheat grain germinates the embryo imbibes water and thecoleoptile cells begin to expand. This expansion process can be stimulatedin excised young coleoptiles by gibberellic acid. Thus in the wheat coleoptilesand also in the young pea stems studied by Purves and Hillman (1958)gibberellins are probably required before the early growth of cell walls canbegin. Other studies which suggest the probable importance of gibberellicacid in early cell-wall growth include those of Wareing (1958), who investi-gated the effects of gibberellic acid and IAA upon cambial activity of shootsof sycamore, poplar, and ash. Gibberellic acid stimulated cell division in thecambium and gave rise to a zone of new tissue consisting of very thin-walledfusiform cells with no lignification (primary cell walls?). Where IAA alonehad been applied, new xylem elements could clearly be discerned on theperiphery of the old wood. However, the new elements did not form a con-tinuous ring around the stem but isolated groups of vessels occurred atintervals. When gibberellic acid and IAA were applied together the cambiumproduced a zone of fully differentiated xylem cells. Thus in these experimentsIAA appears to be essential for xylem differentiation whereas gibberellic acidand possibly IAA are required for primary cell-wall growth. Then there isthe observation of Pecket (i960) that gibberellic acid leads to an increase inroot-hair production on excised pea roots. Root hairs resemble rapidly grow-ing cell walls (i.e. primary cell walls) in respect of their high proportion ofpectic substances and these results, like those above, suggest that gibberellicacid may be important in early cell-wall growth.

The role of kinetin in the sequential growth response is rather an enigmasince although its optimal activity coincides with the period of cell multi-plication, the response that it elicits in the excised coleoptile is one of cell



elongation and not cell division (Wright, 1961a). If one of the results ofkinetin action is to cause a release of energy by the breakdown of food reserves(Wright, 1963), then it is conceivable that in the excised coleoptile this energyis channelled into the cell-elongation process, whereas in the intact coleoptileit might be used in cell division in the presence of some other factor or factors(these we should need to assume diffuse from the coleoptile into the mediumwhen it is excised). The age at which coleoptiles give the maximum kinetinresponse was found to be altered quite markedly by the germination tem-perature (Fig. 3a and b). Haber and Luippold (i960) who have investigatedthe processes of cell elongation and cell division in the root tips of germinatinglettuce seed have shown that a low temperature of germination (io° C) causescell elongation to precede cell division, whereas, at a higher temperature(260 C) cell division and cell elongation take place together. These findingsmay be applicable to the wheat coleoptile and explain the shift in the optimalkinetin response when the growing temperature is changed. At the lowertemperature (22-5° C) cell elongation may precede cell division, whereas atthe higher temperature (25 ° C) the two processes may overlap.

The experiments detailed in Table 1 suggested that substances diffusingfrom the primary leaves within the excised whole coleoptiles did not interactwith gibberellic acid, kinetin, or IAA during the period of treatment in theklinostat. When the primary leaves were absent from the 66-hour coleoptilesections (Table 1, treatments ii and iii) there was a general lowering of thegrowth response. This was probably due to unavoidable damage to the coleop-tile sections when the leaves were ejected. The effect of increasing thenumber of cut surfaces by cutting the older coleoptiles in two (60-hour,Table 2) decreased the growth response to all three growth regulators butin particular the response to kinetin (per cent increment over control length).The results suggested that the low growth response produced by gibberellicacid and kinetin in 60-hour coleoptiles was probably not attributable to anydifficulty in the rate of uptake of these substances into the older coleoptiles.

When the original paper describing the sequential growth response of thewheat coleoptile to gibberellic acid, kinetin and IAA was published in 1961there were several reports in the literature of the isolation of naturally occur-ring kinins from rapidly dividing plant tissues suggesting kinins could beconcerned in controlling normal plant growth (some recent references include:Zwar and Skoog, 1963; Letham, 1964; Maia, 1964; Powell and Pratt, 1964;Zwar et al., 1964). This view has been strengthened recently by the identifica-tion of the kinin of sweet-corn kernels as 6-(4:hydroxy-3-methylbut-2-enyl)-aminopurine (Letham et al., 1964; Shaw and Wilson, 1964) and bythe studies in which kinin-like substances, capable of delaying leaf senescence,have been shown to be present in the bleeding sap of vines (Loeffler and vanOverbeek, 1964) and in root exudates of sunflower seedlings (Kende, 1964).Furthermore, Osborne and Black (1964) have reported the basipetally polartransport of benzyladenine through sections of young petioles of bean, therate of transport being increased twofold in the presence of IAA. Thus it


in the Wheat Coleoptile (Triticum vulgare). II 175

seems probable that naturally occurring kinins may be transported in plantslike true hormones and synthesized in the root rather like the caulocalinespostulated by Went (1938), except that kinins would appear to have widerfunctions than those ascribed to caulocalines.

As can be seen from Fig. ib there is a considerable spread and overlap ofthe growth response to the three growth regulators with coleoptile age whichsuggest that the orderly growth and cellular differentiation of the wheatcoleoptile is determined by a sequence of growth substance mixtures evershifting in composition (Wright, 1961a). It is intended to examine the effectof mixtures of gibberellic acid, kinetin, and IAA on coleoptile growth.

ACKNOWLEDGEMENTS

The author wishes to thank Professor R. L. Wain, F.R.S. for his supportand encouragement of this work and Mr. P. Cozens and Miss C. Johns fortheir painstaking technical assistance.

L I T E R A T U R E C I T E D

HABER, A. H., and LUIPPOLD, H. J., i960. Separation of Mechanisms Initiating Cell Divisionand Cell Expansion in Lettuce Seed Germination. Plant Physiol. 35, 168—73.

KENDE, H., 1964. Preservation of Chlorophyll in Leaf Sections by Substances Obtainedfrom Root Exudates. Science, 145, 1066—7.

LETHAM, D. S., 1964. Isolation of a Kinin from Plum Fruitlets and Other Tissues. Regu-lateurs Naturels de la Croissance Vegetale. Centre National de la Recherche Scientifique,109-19.

, SHANNON, J. S., and MCDONALD, I. R., 1964. The Structure of Zeatin, a FactorInducing Cell Division. Proc. Chem. Soc. 230—1.

LOEFFLER, J. E., and VAN OVERBEEK, J., 1964. Kinin Activity in Coconut Milk. Regulateursnaturels de la croissance vegetale. Centre National de la Recherche Scientifique, 77-83.

MAIA, E., 1964. Isolement d'un facteur de division cellulaire a partir du jus de tomate.Ibid. 103-9.

OSBORNE, D., and BLACK, M. K., 1964. Polar Transport of a Kinin, Benzyladenine. Nature,London, 201, 97.

PECKET, R. C., i960. Effects of Gibberellic Acid on Excised Pea Roots. Ibid. 185, 114-15.POWELL, L. E., and PRATT, C , 1964. Kinins in the Embryo and Endosperm of Prunus

persica. Ibid. 204, 602.PURVES, W. K., and HILLMAN, W. S., 1958. Response of Pea Sections to Indoleacetic Acid,

Gibberellic Acid, and Sucrose as Affected by Length and Distance from Apex. Physiol.Plantarum, I I , 29-35.

SHAW, G., and WILSON, D. V., 1964. A Synthesis of Zeatin. Proc. Chem. Soc. 231.VAN OVERBEEK, J., 1962. Endogenous Regulators of Fruit Growth. Proc. of Campbell Soup

Company Plant Science Symposium, 37-58.WAREING, P. F., 1958. Interaction between Indole-acetic Acid and Gibberellic Acid in

Cambial Activity. Nature, London, 181, 1744-5.WENT, F. W., 1938. Specific Factors other than Auxin Affecting Growth and Root Forma-

tion. Plant Physiol. 13, 55-80.WRIGHT, S. T. C , 1961a. A Sequential Growth Response to Gibberellic Acid, Kinetin and

Indolyl-3-acetic Acid in the Wheat Coleoptile {Triticum vulgare). Nature, London, 190,699-700.19616. Growth and Cellular Differentiation in the Wheat Coleoptile (Triticum vulgare).

I. Estimation of Cell Number, Cell Volume and certain Nitrogenous Constituents.J. Exp. Bot. 12, 303-18.



WRIGHT, S. T. C , 1963. Cellular Differentiation at the Molecular Level with SpecialReference to Proteins. Symp. Soc. exp. Biol. 17, 18—39.

ZWAR, J. A., and SKOOG, F., 1963. Promotion of Cell Division by Extracts from Pea Seedlings.Aust.J. Biol. Sci. 16, 129-39., BRUCE, M. I., BOTTOMLEY, W., and KEFFORD, N. P., 1964. A Comparison of theKinins of Apple and Coconut Milk and of the Modifications Produced by PurificationProcedures. Regulateurs Naturels de la Croissance Vegetale. Centre National de laRecherche Scientifique, 123-30.


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