DAYS AFTER GERMINATION OF VEGETATIVE BUDS TO MATURITY

THE NATURAL AUXINS OF THE SUGAR CANE - ACIDIC, BASIC, AND NEUTRAL GROWTH SUBSTANCES IN ROOTS AND SHOOTS FROM TWELVE DAYS AFTER GERMINATION OF VEGETATIVE BUDS TO MATURITY H. G. CUTLER and A. J. VLITOS Central Agricultural Research Station for Caroni Ltd. and Ste. Madeleine Sugar Co., Ltd., Carapichaima, Trinidad (Presented by Dr. A. J. Vlitos) INTRODUCTION The naturally occurring plant-growth substances in sugar cane at different stages in development have never been fully described. BRANDES and VAN OVERBEEK~ found both 'free' and 'bound' auxin in sugar cane nodes. They noted that 'free' auxin content was depressed in stems which had been heated in water at 50' C for 20 minutes. Loss of apical dominance and improved germination of lateral buds along the axis of the stem were associated with heat treatment. The 'auxin' of BRANDES and VAN OVERBEEK probably represented a mixture of several growth substances, since the individual components of the ether extracts were not separated prior to bioassay; nevertheless their study represents an elegant demonstration of the role of naturallyoccurring growth substances in the development of sugar cane. ENGARD and LARSEN reported that the predominant auxin in sub-apical stem tissues of sugar cane is an acid with chemical and biological properties resembling those of indole-3-acetic acid (IAA). They also mentioned the occurrence of a neutral auxin, which they assumed to be indole-3-acetaldehyde. More recently7 it has been found that the major acidic auxin in the true seed of sugar cane is probably IAA. The present study deals with a paper chromatographic and biological analysis of the growth substances occurring in sugar cane roots and shoots at various stages of development. Extraction and chromatogra$hy MATERIALS AND METHODS Roots and shoots of the sugar cane variety B. 41227 originating from three-bud vegetative setts were harvested 12 days after planting and at 12-day intervals thereafter, for the first four months of growth. From four months of age to maturity (i.e. after floral initiation) the harvests were made monthly. The tissues collected were immediately frozen and ground in ice-cold absolute ethanolin a Waringblendor. The ethanolic breis were stored overnight at -5' C and then filtered. The ethanol extracts were concentrated to aqueous residues. Acidic, neutral, and basic growth substances were then extracted from the aqueous fractions with diethyl ether, using, with slight modification, the method of KEFFORD 4. The ether was removed and the small amount of the aqueous solution remaining (about 1.0 ml) was re-extracted with five times its volume of anhydrous diethyl ether and this ether was then removed and the residue dissolved in absolute ethanol. All evaporations were carried out under vacuum.

SGRICULTURE The ethanol solutions, presumably containing the acidic, basic, and neutral growth substances, were spotted on to Whatman No. I filter paper and chromatographed in an ascending-descending direction in isopropanol-ammonia-water (80 : 5 : 15, v/v/v). Equivalent dry weights of tissue were represented on each chromatogram; an average of 1222 mg of shoot tissue and 437 mg of root tissue being Bioassay of paper chromatograms The developed chromatograms were cut into ten equal strips, from the starting line to the solvent front. Each of the paper strips was numbered and placed in a test tube containing two ml of a phosphate-citrate buffer (ph 5.6) plus two per cent sucrosee. Ten first-internode sections of Avena (var. Missouri-0-205) were floated in each test tube and rotated at & r.p.m. for 14 hours. The Avena sections were measured by projecting their image (X 3) on a flat-table surface by means of a photographic enlarger and any curved sections measured using a flexible rule; all data were analyzed statistically. Each time a bioassay was run, a control experiment with synthetic IAA at 1.0 p.p.m. was included to indicate whether the Avena sections were responsive to growth regulators and whether the bioassay was functioning as TABLE I Rf VALUES OF THE ACIDIC, BASIC, AND NEUTRAL PLANT GROWTH SUBSTANCES OCCURRING IN SUGAR CANE (SHOOTS AND ROOTS) IN VARIOUS STAGES OF DEVELOPMENT (SOLVENT: ISOPROPANOL- AMMONIA-WATER, 80/5/15) Roots I2 0.5-0.7 0.2-0.3 60 0.5-0.6 - - 0.0-0.1 0.0-O.I* 0.0-0.1 0.9-I.O* 0.6-0.7* * Growth inhibitors

I H. G. CUTLER, A. J. VLITOS 34I expected. Bioassays were carried out in a room provided with subdued green light" and maintained at a constant temperature of 72" F f.3". Results were plotted in histograms with the ordinate showing final mean growth (X 3) for ten Avena firstinternode sections and the abscissa representing the Rf value with activity (or inactivity) in the bioassay. Only highly significant (.OI level) difference~ were considered valid. These were plotted as darkened bars in the histograms. Growth curves At each harvest, the heights of the sugar cane plants were recorded. The data were thus plotted at 12-day intervals up to four months of age and at monthly intervals thereafter. Tillers began to develop between 72-84 days after germination. The average heights of tillers were recorded separately from heights of the main stem. EXPERIMENTAL RESULTS The growth-promoting and inhibitory substances in the acidic, basic and neutral fractions in shoots and roots of sugar cane from 12 days to maturity are presented in Table I and the degree of activity at different Rf values of the acidic fractions is illustrated in Figs. I and 2. Identification. of the acidic growth $ronzoter The predominant growth substance in sugar cane during its growth cycle is an acid, running at Rf 0.5 to 0.6 on paper chromatograms developed in isopropanolammonia-water (80: 5 : 15). When the acidic growth promotor was co-chromatographed with synthetic IAA in a number of solvents, the Rf values of the two compounds were almost identical (Fig. 3). In a neutral solvent (n-butanol-ethanol-water, 4 : I : I) and in an acidic solvent (n-butanol-acetic acid-water, 60 : 15 : 25) the Rf values of synthetic IAA and the acidic growth promotor were extremely close. 'Tailing' occurred in the acidic solvent as a result of overloading. In pyridineammonia (80: 20) some spreading also occurred, but the Rf value of the acidic growth promoter could not be sharply differentiated from that of synthetic IAA. Further studies, employing a solvent of n-hexane saturated with water as described by LARSEN =, confirmed that the acidic growth promoter could not be differentiated from the synthetic IAA, both substances giving an Rf value of 0.0 in the hexane solvent. Colorimetric tests indicated that the acidic growth promoter contained an indole ring. Positive colours (bluish-purple) were obtained with sprays of j-dimethylaminocinnamaldehyde on chromatograms developed with Whatman No. 3 MM paper in isopropanol-ammonia-water (80: 5 : 15). The colours developed at an Rf value which corresponded to the areas of biological activily (0.5 to 0.6), and with those of synthetic IAA, co-chromatographed with tissue extracts containing the acidic growth promoter. There is therefore presumptive evidence that the major auxin in sugar cane is in fact IAA. One point in technique worthy of mention is that when co-chromatographing synthetic IAA in mixtures with the tissue extracts we found a depression of Rf value for the IAA as compared to the Rf value for IAA chromatographed singly. This is

342 AGRICULTURE 22-21 - 20-12DAYS 24 DAYS 36 DAYS I9-18 - 14 13-4- --r~ L - E 22- E 21-.= 20 84 DAYS 96 DAYS I08 DAYS - 5 I9 ; 187: : A- s 14-182 DAYS 312 DAYS Fig. I. Rf values of acidic, ether-soluble growth substances isolated from 12- to 312-days old shoots of sugar cane plants (solvent: isopropanol-ammonia-water, 80 : 5 : 15, v/v/v). particularly so when large quantities of the extracts are applied to Whatman No. I paper, for example when one wishes to load enough of the extract on the paper to determine a colour reaction. It was possible to avoid the depression in Rf value by using Whatman No. 3 MM paper. The absorptive capacity of the heavier paper permitted much greater quantities of tissue extract to be applied without distorting the Rf value of the IAA which had previously been added to the extract.

H. G. CUTLER, A. J. VLITOS 343 21 ;;!:-A- - 20 - I2 DAYS 24 DAYS 36 DAYS A wnw 15-14 - 13-21 -., E 16-15 - 2 I4 - x 13- E 21- c 20-84DAYS. - 96 DAYS lob DAYS r - 19 * 3 IB- 9 17- U '6- c 15 0 14 -Am- - 13- - 0 c 21 19 18 L- L- 15 14 13 1-L I20 DAYS 151 DAYS 182 DAYS - L- - 0 21 - Rf value-2 20-213 DAYS 312 DAYS 19- I8-17 - :, -- h- 14-13 - - O Rf valuelo - O Rf ~ a l u e 2 O Fig. 2. Rf values of acidic, ether-soluble growth substances isolated from 12- to 312-days old roots of sugar cane plants (solvent: isopropanol-ammonia-water, 80 : 5 : 15, v/v/v). Growth in relation to 'IAA' content of sugar cane roots during development The growth of sugar cane plants from 12 days up to maturity is depicted in histogram form in Fig. 4. Growth was most rapid between o and 36 days. If one compares relative quantities of the acidic growth promoter in the roots from 12 days to four months it is seen that where a relatively large increment of growth in shoots preceded the date of extraction, the relative amounts of the growth promoter in the roots were decreased. Less activity was detected in roots extracted at 24 and 36 days of age than in subsequent extractions up to 72 days. From 72 days of age onwards, coinciding with the initiation and development of tillers, there was a drop in the levels of 'IAA' in the roots. The relative quantities of 'IAA' in roots appeared to be inversely proportional to the preceding increment of growth in shoots and/or tillers.

H. G. CUTLER, A. J. VLITOS 345 Aqe in days at Harvest Fig. 4. Growth of sugar cane shoots from 12 to 312 days expressed as height in centimetres Double bars from 84 to 312 days represent growth of main shoot (upper bars) and of tillers (lower bars). DISCUSSION The predominant growth substance in sugar cane roots and shoots is an ether-soluble acid with chromatographic and colorimetric properties similar to those of IAA. Extracts of roots and shoots from 12 days after germination of vegetative buds and at 12-days intervals up to maturity contained the compound in varying amounts. Several other growth substances were also detected at different stages in ontogeny. Three different neutral growth promoters occurred in extracts of shoots, one of these was detected in 12-day old shoots, a second in 84-day old shoots, and a third in 96-day old tissues. A basic growth promoter was detected in extracts of 24-day old shoots, and another in 108-day old shoot tissues. Roots harvested at 60 days contained, in addition to 'IAA', a second acidic growth promotor whilst a neutral growth substance was found at six months. Two basic growth promoters occurred in five-month old roots. A number of growth inhibitors were detected in both shoots and roots throughout the growth cycle, but most of these were confined to the roots during early study of development. One interesting feature of the study was the lack of either tryptophane or skatole in either root or shoot extracts as determined by colorimetric or biological methods. Skatole has been reported to be present in seed of sugar cane7. None of the auxiliary growth promoters were present in as large a quantity nor were they as active as the major acidic auxin. They did not occur as consistently as

AGRICULTURE 1 % the latter during each stage in development nor were they related noticeably with growth of sugar cane. On the other hand there was a striking relationship between the amount of 'IAA' in roots and the preceding growth rate in above-ground portions of the plant. Whether or not the roots act as a 'reservoir' of supra-optimal quantities of 'IAA', releasing the auxin to the developing shoots as required, has not been determined. It is of interest however that the drop in levels of 'IAA' in roots coincided with the development of the main stem and tillers, which also suggests that some relationship may exist between the supply of 'IAA' in roots and the subsequent growth of sugar cane tillers. There are three possible explanations for the observed relationship : (I) either the auxin is synthesized in the stems, translocated and stored in the roots for use in periods of rapid growth (in the stems and roots) ; (2) the auxin is manufactured in the roots and translocated to the stems, and (3) the auxin is manufactured at both sites and subsequently utilized at both sites. The complexity of the 'auxin' system in sugar cane was demonstrated by the many different growth substances that were detected during ontogeny. Not only did different growth substances occur at different stages of development, but different profiles were obtained from roots and shoots of plants of the same age! From a physiological point of view it would be of interest to ascertain whether each of the many growth substances detected at the different stages in development is required for a specific physiological function. The Avena first-internode bioassay merely demonstrates that a substance is active in promoting cellular elongation and that there is a possibility that a similar effect can be induced in the intact plant. Future studies on auxin systems in other genera may have to consider more fully the many changes in growth substance profiles during ontogeny, as well as the various types of growth substances occurring in the different organs on the same plant. The evidence presented here is heavily in favour of the idea of a multi-growth substance system in sugar cane; a much more complex picture than has heretofore been described. SUMMARY The growth substances occurring in roots and shoots of sugar cane at various stages in its development have been characterized by a combination of paper chromatography, bioassays, and colorimetric reactions. From 12 days after germination of the vegetative bud to maturity the predominant growth promoter is an acid with properties similar to those of IAA. A number of other growth promoters and inhibitors also occurred in both the roots and shoots at various stages - in development. Relatively less 'IAA' was detected in roots if, prior to extraction, there had been a large increment in growth of the shoots, or of the tillers. REFERENCES 1 BRANDES, E. W. and VAN OVERBEEK, J., 1948. Auxin relations in hot-water treated sugar cane stems. J. ag~ic. Res. 77 : 223-38. a ENGARD, C. J. and LARSEN, N., 1951. Auxin investigations in sugar cane. Re+. Hawaii. agric. Ex+. Sta., 1948-1950 : 139-40. HARLEY-MASON, J. and ARCHER, A. A. P. G., 1958. Use of +-dimethylaminocinnamaldehyde as a spray reagent for indole derivatives on paper chromatograms. Biochem. J. 69 : 60P. KEFFORD, N. P., 1955. The growth substances separated from plant extracts by chiomatography. J. ex+. Bot., 6 : 129-51.

F T. CHINLOY, T. C. E. WELLS, N. J. CHIN, J. L. RAMSAY 6 LARSEN, P. and AASHEIM, T., 1961. The occurrence of indole-3-acetaldehyde in certain plant extracts. 4th Int. Conf. on PCant Growth Regulatzon : 43-55. 6 NITSCH, J. P. and NITSCH, C., 1956 Studies on growth of coleoptile and first-internode sections. A new, sensitive straight-growth test for auxins. Plant Physiol., 31 : 94-111. 7 VLITOS, A. J, arid CUTLER, H. G., (1960). The natural auxins of the sugar cane. I: A paper- chromatographic separation of the growth factors present in true seed. Proc. Brit. W. Ind. Sug. Tech., 1960: 113-127. -. DISCUSSIONS C. J. MONGELARD (Mauritius): The type of tissue from which extraction is made is certainly different in 12-day old and 312-day old shoots; is the picture obtained comparable? A. J. VLITOS (Trinidad): In each case the dry weight of tissue used for chromatograms was the same. MR. MONGELARD; Have you tried the specific test for IAA, i.e. the perchloric acid - ferric chloride reagent of Gordon and Weber? DR. VLITOS: We have tried many test reagents and are satisfied from the Rf values and colourimetric tests that the material is almost certainly IAA. POSSIBILITY OF LONG-FURROW IRRIGATION UNDER HEAVY CLAY SOIL CONDITIONS T. CHINLOY, T. C. E. WELLS, N. J. CHIN and J. L. RAMSAY The Sugar Manufacturers' Association (of Jamaica) Ltd.; Bernard Lodge Sugar Co.; The West Indies Sugar Co. Ltd.; Sevens Ltd.; Jamaica (Presented by Mr. N. J. Chin) INTRODUCTION Surface irrigation is practised on about 50% of the cane-growing area of sugar estates % in Jamaica at a cost ranging up to 20 per acre per annum for water and its application. It is of the greatest importance that water be efficiently used and at optimum rates of application; and in these considerations, the uniformity of distribution over the surface of the land, the quantity of soil recharge within the root range of sugar cane, and the wastage by runoff or deep penetration, are important. Formerly, sugar cane culture and methods of reaping and infield transport permitted the use of a system which became general and which is illustrated in Fig. I. The system consists of a 'head' main canal which feeds 'infield mains' running at right angles and parallel with the cane rows. 'Twigs' or smaller channels, spaced 11 to 22 yards apart, lead water across the cane rows from the 'infield mains'. The comparatively close spacing of the 'twigs' caters for a fair degree of non uniformity of land surface; and on heavy soils the 'twigs' and 'infield mains' may be sunk to 18" depth to act as runoff drains. However, the extent to which such 'drains' bring about any effective draining of the subsoil mass is in doubt. The system can distribute water effectively over fairly uneven land but is very difficult to inspect and supervise, except from the air; the amount of manual 'working' of the water is high, i.e. the system is not easily yendered automatic, and the wastage of water by runoff is sometimes high, particularly at night. Aerial inspection shows that during periods of water