New Phytol. {ig()) S, ioiyio22. EFFECTS OF GIBBERELLIC ACID ON INTERNODE GROWTH AND STARCH CONTENTS OF EUCALYPTUS CAMALDULENSIS SEEDLINGS BY E. P. BACHELARD Department of Forestry, Australian National University, Canberra, A.C.T., Australia {Received 23 April 199) SUMMARY Effects of gibberellic acid (i and /ig/plant) on internode extension and cell activity in Eucalyptus camaldulensis seedlings varied with internode position. The relatively small increased ejrtension of lower internodes resulted from cell elongation whereas the progressively increasing growth in upper internodes was caused primarily by cell division. Transverse cell divisions induced by gibberellic acid seemed to require an active apical meristem. Changes in internode diameter growth (increased in lower internodes, decreased in upper) due to gibberellic acid were the result of changes in cell division. The major response to gibberellic acid seems to be an increase in transverse cell divisions and does not seem to be due to competition between meristems for the available food reserves. INTRODUCTION Modifications in the normal pattern of eucalypt seedling growth, following seed treatments with gibberellic acid have been attributed to increased activity of the main shootapical meristem at the expense of other meristems (Bachelard, 198). Gibberellic aeid was thought to break down food reserves (particularly starch) making assimilable substrates more readily available to an active meristem. In this investigation these possible effects of gibberellic acid were examined. MATERIAL AND METHODS Seeds of Eucalyptus camaldulensis Dehn. were sown in a seed box and after 5 weeks the seedlings were transplanted into veneer tubes ( in. high x 2 in. diameter) containing a i: i mixture of silty loam and coarse gravel. The seedlings were grown in a glasshouse, equipped with heaters and coolers to avoid temperature extremes, under 1hour days (normal day lengths supplemented with fluorescent and incandescent lights). After 2 months, when most seedlings carried three or four developed leaf pairs, the ninety most uniform seedlings were treated with gibberellic acid by applying jaoi the appropriate solution to the middle of one leaf of the third leaf pair. The solutions, which all contained 1% detergent, were made up such that o, i or jug GA/plant was applied to each of thirty seedlings. After the solutions had dried (2 hours after application), ten seedlings within each GA treatment were retained intact (Intact), ten had their main shoot apical meristem re 17
ioi8 E. P. BACHELARD moved (RA) and ten, in addition to having their main shoot apical meristem removed, had all subsequent axillary shoots removed as they appeared (RA I). Internode lengths of the main shoot axis were measured at weekly intervals using a millimetre rule. At the conclusion of the experiment, 5 weeks after treatment, the average diameter at the midpoint of each internode was obtained using a micrometer gauge, by averaging two diameter measurements taken at right angles. At completion of the experiment, three seedlings from each treatment were harvested (a) Intact RA RA + 2 3 4 5 0 1 3 4 5 0 3 4 5 (b) Intact RA RA + I 2 3 4 5 0 1 2 3 4 5 I 2 3 4 5 Time (weeks) Fig. I. Growth of (a) internode 2 and (b) internode 3 in Eucalyptus camatdutensis seedlings receiving different apical treatments (see text) after treatment with o (o), i ( ) and (D) //g GA/pIant applied at i week. (Each mean based on ten observations and standard errors of total growth shown.) and the internodes fixed in FAA prior to embedding in paraflin wax, sectioning and mounting on shdes using standard techniques (Jensen, 192). Sections were stained with toluidine blue or IKI and photographed through a microscope. Cell lengths and starch contents were measured or observed from the photographs. RESULTS Seedling growth Overall, GA greatly increased internode length, the response increasing progressively with internode number (Figs. 13, Table i). The relative lack of an additional effect at the higher GA concentration suggests the system is almost saturated at a concentration of I fig GA/plant.
Effects of gibberellic acid on Eucalyptus 19 In all seedlings, GA bad no effect on tbe length of internode 2, and increased the length of internode 3 by 1. times. In Intact seedlings, the effects of GA increased progressively from an increase of about 3 times for internode 4 to about 13 times for internode. Although the data are limited, owing to the absence of seedlings with more than four internodes at treatment, removal of the apex appeared to limit the effects of GA on internode extension. Internode 4 in RAtreated seedlings increased only by 1. times compared with 3.1 times in Intact seedlings (Table i). s o <3 38 _ 3 34 32 30 28 2 24 22 20 18 1 14 12 8 4 2 Intoct 1 t / / il I t 1 ^ ' ; / * : / T ~v / ^ 1 1 1 1 1 I 2 3 4 5 0 2 3 4 5 2 3 4 5 Time (weeks) Fig. 2. Growth of internode 4 in Eucalyptus camaldulensis seedlings receiving different apical treatments (see text) after treatment with o (O), i ( ) and ( ) ug GA/pIant applied at i week. (Each mean based on ten observations (except where shown in parentheses) and standard errors of total growth shown.) Table treated I. Ratios plants to Seedling treatmenit 2 Intact 0.8 RA 0.8 RA + 13 RA of internode lengths internode lengths in plants Internode 3 4 5 1. 3.1.1 1. 1. 1. y in GAuntreated Before examining the effects of treatment on cell lengths it was necessary to determine whether cell lengths varied with position in the internode. Comparison of pith cell lengths from the top and basal thirds of internodes 3 and 5 from Intact seedlings showed the basal cells to be consistently and significantly longer than cells at the top of the internode (Table 2). These data also showed comparable cells in internodes 3 and 5 to be of much the same length, and that GA treatment increased cell lengths by about 1. times. Comparison of cell lengths from the top third of internode 3 showed no effect of 133
IO2O E. P. BACHELARD removal of apical meristems and, in all cases, GA treatment again increased cell length by an average of 1.52 times (Table 3). The diameters of internodes 2 and 3 were increased by GA in all treatments and the 40 38 3 34 32 30 28 2 24 "E 22 ~ 20 18 1 14 12 8 4 2 (a) 8 4 2 8 4 2 (b) If ^ 5 I 2 3 5 (c) 5 3 4 5 Time (weeks) Fig. 3. Growth of (a) internode 5, (b) internode and (c) internode 7 in Intact Eucalyptus camaldulensis seedlings after treatment with o (O), i ( ) and (D) n% GA/plant applied at I week. (Each mean based on ten observations and standard errors of total growth shown.) Table 2. Ejfects of gibberellic acid on pith cell lengths from the top and basal thirds of Eucalyptus camaldulensis internodes Treatment Internode Internode no. (/ig GA/plant) position 3 5 0 Top 39i±iS 27.2± I.O Base 47.3 ±2.2 47.7 + 2.1 I Top 497 ±19 443 ±14 Base 92.9±39 4. ±2.2 Top 5.5 + 2. 47.9 + 2.5 Base 79 ±33 7.9 ±2.9 Average cell length increase due to GA treatment X 1.7 X 1. Each mean and standard error based on measurement of fifty cells. response was much the same in the presence or absence of apical meristems (Fig. 4). By internode 4, in Intact seedlings, the diameter of GAtreated seedlings started to decrease sharply until, by internode 5, the diameter of treated seedlings was less than in un
Effects of gibberellic acid on Eucalyptus 21 treated seedlings. The diameters of internode 4 of seedlings from which the apical meristems were removed and which received i /ig GA/plant did not decline as sharply as in Intact seedlings but there are too few data to confirm this. Measurements of the size of pith cells in transverse sections from internodes 3 and 5 in Intact seedlings showed no differences due to GA indicating the effects of GA on internode diameter were caused by effects on cell division. Table 3. Effects of gibberellic acid and apical treatment of seedlings on pith cell lengths {fi) from the top third of internode 3 of Eucalyptus camaldulensis seedlings Treatment ^g GA/plant) o I Average cell length increase due to GA treatment Intact 391 ±15 497 ±19 5.5 + 2. X 1.5 Apical treatment Apex removed Apex and subsequent (RA) axillary shoots removed (RA + ) 374 ±14 32.8 + 1.2 3.1 ±2.4 2.7 ±3.1 4.4 + 2. 9.1 + 2.9 X1.5 Each mean and standard error based on measurement of fifty cells. 1 15 14 13 12 E Intact RA + fe I 09 ^ 08 0 7 0 05 Internode no. Fig. 4. Internode diameters in Eucalyptus camaldulensis seedlings receiving different apical treatments (see text) 5 weeks after treatment with o (O), i ( ) and (D) //g GA/plant. (Each mean based on ten observations (except where shoviti in parentheses) and standard errors shown.) Starch contents Effects of treatments on residual starch contents were examined in transverse and longitudinal sections from internodes 2, 3 and 5 by staining with IKI solution. Representative transverse sections are shown in Plate i. Generally, starch concentrations were considerably reduced by ^g GA but not by i ^g. Since i ug GA was about as effective as /ig in the growth response, there is no evidence that growth depended on starch breakdown.
22 E. P. BACHELARD DISCUSSION The effects of GA on internode extension growth and on cell activity varied with position of the internode in the seedling. Internode 2 was unaffected by treatment, and the increased extension growth of internode 3 could be accounted for completely by increased cell elongation. Presumably, the cells in internode 2 had ceased both cell division and cell elongation at the time of treatment whilst the cells in internode 3 still retained a capacity for cell elongation only. In higher internodes in Intact seedlings, GA increased both cell elongation and transverse cell divisions, the latter becoming progressively more important in internode extension growth. GAinduced elongation, by transverse cell divisions, in internode 4 of some Intact seedlings occurred some distance below the apical meristem. This was a result which supports earlier reports of the importance of subapical cell divisions in GAinduced shoot elongation (Sachs, Bretz and Lang, 1959). The relative lack of effect of GA on the elongation of internode 4 in seedlings from which apical meristems were removed suggests GA requires some other factor derived from the apex to stimulate cell division. Internode diameters were also altered by treatment with GA and, in all cases, the effects were due to changes in longitudinal cell divisions. In Intact seedlings, there was some indication of an inverse relationship between transverse and longitudinal cell divisions induced by GA. In the lower internodes (2 and 3), in which transverse cell divisions were unaltered by GA, longitudinal cell divisions increased, whilst in the higher internodes (4 and 5) increased transverse cell divisions were accompanied by an unaltered or decreased number of logitudinal cell divisions. The absence of a marked decline in diameter growth of internode 4 in RAtreated seedlings (an internode in which the GAinduced extension growth appeared due to cell elongation alone) offers some support for this inverse relationship. Diameter growth was affected only slightly by GA relative to extension growth confirming other findings that by far the majority of cell divisions induced by GA give rise to transverse walls (Sachs et al., 1959). Removal of apical meristems did not markedly infiuence the effects of GA on diameter growth, and the starch reserves were never obviously reduced by GA at least at a concentration of I yug/plant. These results indicate that the response to GA is not caused simply by assimilable substrates being made more readily available to an active meristem. REFERENCES BACHELARD, E. P. (198). Effects of seed treatments with gibberellic acid on subsequent growth of some eucalypt seedlings. New Phytol., 7, 595. JENSEN, W. A. (192). Botanical Histochemistry. Principles and Practice. London. SACHS, R. M., BRETZ, C. F. & LANG, A. (1959). Shoot histogenesis: The early effects of gibberellin upon stem elongation in two rosette plants. Am. J. Bot., 4, 37.
THE NEW PHYTOLOGIST, 8, 4 PLATE I Starch contents in transverse sections from the base of internodes of Intact Eucalyptus camaldulensis seedlings, 5 weeks after treatment with gibberellic acid, (ac) Internode 3 at o, I and //g GA/plant respectively, (df) Internode 5 at o, i and //g GA/plant respectively. E. P. BACHELARD OF GIBBERELLIC ACID ON EUCALYPTUS (facing page 22)