AN IAA-INDUCED STIMULATION OF GROWTH OF ROOTS OF VICIA FABA PREVIOUSLY TREATED WITH COLCHICINE

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1 AN IAA-INDUCED STIMULATION OF GROWTH OF ROOTS OF VICIA FABA PREVIOUSLY TREATED WITH COLCHICINE BY D. DAVIDSON*, R. D. MACLEOD AND I A N E T TAYLORt Department of Botany, University of St. Andrews, Scotland (Received 4 December 1964) SUMMARY Roots of Viciafaha were treated with a 0.02 'o solution of colchicine for 2 hours. Some roots were also treated, 22 hours later, with lo^e M IAA for i day. All beans were grown on for a further 6 days and the total growth of primary and lateral roots was determined. Primary roots treated with colchicine and then IAA showed significantly greater growth than roots treated only with colchicine. Lateral roots produced by the treated primary roots showed significantly less growth when treated with colchicine and IAA than when treated only with colchicine. Primary- roots treated with colchicine and IAA had fewer polyploid cells, in division, than roots treated only with colchicine, indicating that recovery of the diploid condition is related to the rate of root growth. IAA treatment does not produce an immediate stimulation of growth; no effect was seen for 3 days. Its effect appears to be one of facilitating the normal mechanisms by which an apical meristem recovers from a colchicine treatment. The stimulation of root growth by concentrations of IAA that are normally inhibitory leads us to conclude that one result of a treatment with colchicine is a reduction in the level of natural auxin in a root. INTRODUCTION Longitudinal growth of roots can be arrested by treatment with colchicine solutions. Initially this inhibition is due to the failure of the cells in the region of elongation to expand linearly, although they show isodiametric expansion (Hawkes, 1942) and so produce an apical swelling, the C-tumour (Levan, 1938; Eigsti and Dustin, 1955). Longitudinal growth continues to be inhibited for several days, although, however, regeneration will occur if the strength of the solutions used is not above certain levels (Levan, 1938; Witkus and Berger, 1950; Davidson, 1961a). In primary roots of Vicia faba regeneration does not occur for from 3 to 7 days after treatment with % colchicine for i or 3 hours (Davidson, 1961a). Lateral roots are also inhibited by colchicine (Davidson, 1964a). Indole-3-acetic acid (IAA), which occurs naturally in root apices (Bennet-Clark and Kefford, 1953; Leopold, 1955), also inhibits root growth (Noirfalise, 1940; Beal, 1951). Noirfalise (1940) found that growth of V.faba roots is completely inhibited by IO-^M solutions of IAA, the inhibition persisting for 3 days, and is partially inhibited by 5 X io"^ M IAA. Using sixteen or seventeen beans per treatment, it has been shown that roots exposed to 10-5 jvi ly^ fqj. J (jgy^ 24 hours after treatment with 0.05 % colchicine for 3 hours, produced more new growth than roots treated only with colchicine (Davidson, 19616). * Present address: Biology Department, Western Reserve University, Cleveland, Ohio. t Present address: The Botany School, Oxford. 393

2 394 D. DAVIDSON, R. D. MACLEOD AND JANET TAYLOR It was also found that the same treatment with IAA alone (io-5 M for i day) produced a 56 % inhibition of growth when compared with untreated controls. These results show that the response of roots to IAA changes after treatment with colchicine; solutions of IAA that inhibit growth in normal roots, stimulate growth in colchicine-treated roots. The results to be reported here confirm and extend this observation. MATERIAL AND METHODS Broad beans cv. Sutton's Exhibition Longpod were soaked for 24 hours. The testas were then removed and the beans were germinated in moist sand at 20^ C. After about 60 hours the beans with radicles about 6 cm long were washed and suspended over distilled water for 24 hours. They were then treated with 0.02 % colchicine for 2 hours, washed and grown on. After 22 hours, half the beans were transferred to a solution of IAA for I day. In the first experiment roots were grown in io-5 M IAA. This resulted in the death of almost all the roots. In the repetition, to be described here, the concentration of IAA used was io-s M. All beans were then kept in aerated Hoagland's solution, made with distilled water, at 20 C. The solution was changed every 36 hours. Measurements were made of the length of new growth of primary roots 4 and 6 days after the end of the IAA treatment. Lateral roots were also measured on the sixth day. After the final measurements had been made some roots were fixed in chilled acetic acid-alcohol (1/3,v/v) with one drop of formalin added. These roots were hydrolysed in I N HCl for 7 minutes and stained with Feulgen. Squashes were made of their apical meristems. RESULTS The first visible response to colchicine, the formation of the C-tumour, was seen 12 hours after the end of treatment with colchicine. The swelling continued for a further 24 hours, but was less pronounced in the roots that had, meantime, been placed in IAA, i.e. IAA appeared to inhibit swelling in the C-tumour. Resumption of growth of the primary roots began 3 days after the end of the treatment with IAA. In both groups of beans about half had begun to grow again; 56 % after colchicine and IAA, 50.4 % after colchicine alone. The difference between the two groups does not appear to be significant. Table i. Length of new growth of primary roots 0/Vicia faba 4 and 6 days after treatment with 0.02% colchicine or with colchicine followed by 10-^ M IAA Days after IAA 4 4 Treatment + IAA No. of roots treated Mean length of new growth (mm) 10.5 ± ± 2.6 ^Mean difference J IAA ± ±io-5 >Mean difference J 9-7 Primary roots were measured on the fourth and sixth days after the end of the IAA treatment. Roots that had been treated with IAA showed the greatest growth. The diiterence between the mean growth of beans treated with colchicine or with colchicine and IAA is statistically significant at the level P = (Table i). The differences

3 IAA stimulation of colchicine-treated roots 395 between the means are 3.4 on day 4 and 9.7 on day 6. Thus, treatment with exogenous IAA has increased the growth of primary roots previously exposed to colchicine. The relatively greater growth of roots treated with IAA after colchicine is not due to extensive growth by a few primary roots. After 4 days, 53.7 % of the roots treated with colchicme and IAA had i cm or more new growth and 32.8 % had 1.5 cm or more new growth, compared with 16.6% and 1.5% respectively in roots not treated with IAA. After 6 days, 37.9 % of the roots treated with colchicine and IAA had produced 2 cm or more new gro\\th while of those treated with colchicine alone, only 9.9 % had done so. There appears to be a genuine stimulation by the IAA of root growth. It should also be noted, however, that of the si.xty-seven roots treated with colchicine and IAA, thirteen showed no regeneration compared with four of the sixty-six roots treated only with colchicine (see Discussion). As primary roots of Vicia faba recover from the effects of colchicine the composition of their apical meristems is known to change (Davidson, 1961a, 19646). Squashes of the apical meristems were made, therefore, of five roots from each treatment. The mean percentage of polyploid cells in roots treated with colchicine was 19.3 % (mean length of all roots in sample = 9.3^10.5 mm); in roots that were also treated with IAA (mean length = I9.o±i6.2 mm) there were 11.5 % polyploid cells (Table 2). As roots grow, the composition of their apical meristem changes and the frequency of diploid cells increases. It can be seen that the mixoploid condition in an apical meristem of a root does not prevent root growth. Table 2. Frequencies of diploid and polyploid cells in primary roots of \ici2. faba 6 days after treatment with 0.02 % colchicine or with colchicine followed by io-s M IAA - IAA Cells n Total Mean [, polyploid Lateral roots emerged from the treated primaries and were counted and measured on the sixth day. Beans treated with colchicine alone had 124 laterals with a mean length of cm; those treated with colchicine and IAA produced 164 laterals, mean length cm. The mean lengths of these laterals are significantly different at about P = IAA appears, therefore, to inhibit the growth of lateral roots while stimulating the growth of primary roots. It should be noted that these lateral roots were not visible at the time of treatment with colchicine; they emerged after treatment. They grow from primordia that do not respond to colchicine, i.e. their growth is not inhibited and polyploid cells are not formed in their apical meristems. These primordia, which are apparently resistant to colchicine, have been described elsewhere (Davidson, 1964a). Thus, those laterals whose growth is not inhibited by colchicine are inhibited by a subsequent treatment with IAA; and they are the opposite of primary roots, which are inhibited by colchicine but are stimulated by subsequent treatment with IAA. 19 3

4 396 D. DAVIDSON, R. D. MACLEOD AND JANET TAYLOR DISCUSSION It is clear, from the results reported here, that exposure to io-«m IAA 24 hours after treatment with colchicine stimulates root growth. As io^s M or stronger solutions of IAA are known to inhibit root growth (Noirfalise, 1940; Davidson, 1961^) in normal roots of Vicia faba it also appears that colchicine-treated roots show a lower sensitivity to exogenous IAA. This could be due to either a genuine fall in sensitivity, associated perhaps with an increase in the formation of enzymes such as IAA-peroxidase, or a fall in auxin synthesis within the root. The roots, however, are not uniform in response. It has been seen that thirteen of the sixty-six roots treated with colchicine and IAA failed to regenerate, while only four of sixty-six roots treated with colchicine alone failed so to do. It appears that even after treatment with colchicine and, apparently, a consequent disturbance in the auxin status of the root, io-^ M IAA is still inhibitory for some roots. (In our first experiments, 24 hours in io-s M IAA was inhibitory for all roots.) The response of the roots to IAA, i.e. in showing new growth, is interesting because it is not immediate; it is not apparent till 4 days after treatment. Since IAA-treated primary roots do not recommence growth faster than roots not treated with IAA it would seem that the effect of IAA is not merely one of promoting elongation. IAA may stimulate root growth by an effect on cell division, as can be seen by comparing the composition of the meristems of roots that received different treatments. Primary roots treated with colchicine and then IAA had, after 6 days, 11.9% polyploid cells (Table 2) and the mean length of new growth was mm. Comparable roots, treated with colchicine alone, had 19.3 % polyploid cells and had produced 9.3 ± 10.5 mm new growth. Vicia roots that have been treated with colchicine have mixoploid meristems 1-2 days later but they gradually revert to the diploid condition (Davidson, 1961, 1964a, b), i.e. their meristems show an increasing frequency of diploid cells as the period from the time of colchicine treatment increases. The results reported here show that the rate at which roots revert to the diploid condition is related not only to the length of the recovery period but also to the rate of growth in that period. The lower frequency of polyploid cells in roots treated with IAA following colchicine could be due to a selective inhibition, by IAA, of polyploid cells either during root growth (cf. Torrey, 1959) or during the establishment of the primordium from which regeneration occurs. It seems probable, however, in view of the results both from laterals from the C-tumour (Davidson, 1961a, 1964a) and from decapitated or irradiated roots (Davidson, 1961a) that the lower frequency of polyploid cells in IAA-treated roots is due solely to a stimulation of the normal processes by which mixoploid roots revert to the diploid condition. IAA appears to facilitate the recovery, by the root meristem, of the condition in which it will maintain growth in all its phases, not only in cell expansion. Three lines of evidence indicate how IAA is effective: (i) roots treated with colchicine then IAA do not begin to grow again before roots treated with colchicine alone, (2) the effect of IAA is seen many days after treatment, and (3) IAA-treated roots are longer and have a lower frequency of polyploid cells than roots treated only with colchicine. They lead us to conclude that IAA exerts its stimulatory effect by increasing the rate at which the apical meristem of the primary root recovers from the colchicine treatment and reorganizes a functional growing point. Therefore, one of the factors limiting growth in roots treated with colchicine is a shortage of natural auxin. The evidence indicates that IAA allows the colchicine-treated roots to surmount the difficult period when reorganization is taking place; it appears to facilitate the formation of a new apical meristem

5 IAA stimulation of colchicine-treated roots 397 within the old one, disorganized by colchicine treatment. It also appears that at least some of the roots formed after treatment with L\A are capable of sustaining, over the following few days, higher growth rates than roots not treated with IAA. Roots have been treated with colchicine and IAA in previous investigations. Levine and Lein (1941) and Mangenot (1942) treated roots with IAA before colchicine treatment; their experiments could not, of course, reveal any change in sensitivity to auxin that may occur after treatment with colchicine. Hawkes (1942) treated Allium cepa roots with IAA immediately after treatment with colchicine and noted that the size of C- tumours induced by colchicine was reduced by IAA treatment. Treatments with combined solutions, L\A and colchicine (Duhamet, 1945) revealed that the presence of IAA prevented the inhibition of root growth normally induced by colchicine. The results of Hawkes and Duhamet indicate that two aspects of root growth that are disturbed by colchicine are less markedly upset in the presence of IAA: C-tumour expansion is reduced by LAA and the inhibition of root growth is reduced by IAA. Our results agree with both these observations. The evidence of Hawkes (1942), Duhamet (1945) and that reported here supports the hypothesis that a change in the auxin status of roots occurs following treatment with colchicine. By separating the colchicine and IAA treatments by i day we hoped that tbe change in auxin status would become more marked than in previous experiments. We believe that this happened; for whereas Duhamet increased root growth of colchicine-treated roots with 1.14X \o~^'- M IAA, a concentration that was found to stimulate root growth, we have found a stimulation of root gro\\i;h with io-^ M L\A, a concentration that is normally inhibitory to root growth. The lateral roots produced by tbe primary roots appeared after treatment with colchicine. Although they did not respond to the colchicine (Davidson, 1964^) they were somewhat inhibited by IAA and were shorter than laterals from roots not treated with IAA after the colchicine treatment. Whatever change occurs in primary roots, after colchicine treatment, that enables them to be stimulated by usually inhibitory concentrations of exogenous IAA, it is obvious that the same change does not occur in those laterals whose growth is not inhibited by colchicine. Further observations on the response of lateral roots to IAA with, and without, a previous colchicine treatment, are currently being made in this laboratory. REFERENCES BE.^L, J. M. (1951). Histological responses to growth-regulating substances. Plant Grou-th Substances (Ed. by E. Skoog), pp University of Wisconsin Press. BENNET-CLARK, T. A. & KEFEORD, N. P. (1953). Chromatography of the growth substances in plant extracts. Nature, Lond., 171, 645. DAVIDSON, D. (1961a). Mechanisms of reorganization and repopulation in meristems in roots of Vicia faba. Chromosoma, 12, 484. DAVIDSON, D. (19616). Root growth following treatment with colchicine and IAA. Semi-annual Report, Biology Division, Oak Ridge National Laboratory. DAVIDSON, D. (1964a). A differential response to colchicine of meristems of roots of Vicia faba. Ann. Bot. N.S., 29, 253. DAVIDSON, D. (1964^)). Cytological chimaeras in roots of Vicia faba. Bot. Gas. 126, 149. DUHAMET, L. (1945). Recherches sur l'action de l'heteroauxine et de la colchicine sur la croissance des racines isolees de Lupinus albus. Revue CytoL Cytophysiol. veg., 8, 35. EiGSTi, O. J. & DusTiN, P. (1955). in Agriculture, Medicine, Biology and Chemistry. Iowa State College Press, Ames, Iowa. H.AWKES J. (1942). Some effects of the drug colchicine on cell division, jf. Genet. 44, 11. LEOPOLD A. C. (1955). Auxins and Plant Growth. University of California Press. LEVAN, A. (1938). The effect of colchicine on root mitoses in Allium. Hereditas, Lund., 24, 471.

6 398 D. DAVIDSON, R. D. MACLEOD AND JANET TAYLOR LEVINE, M. & LEIN, J. (1942). The effects of various growth substances on the number and the length of roots of Allium cepa. Am.J. Bot., 28, 163. MANGENOT, G. (1942). ^c?;'on rff/a Co/c/z/cme s»r/m/?aanei (f'^wmm cepa. Hermann, Paris. ^ NoiRFALiSE, A. (1940). Recherches sur le d^veloppement des racines de Viciafaba traitees a l'heteroauxine. Cellule, 48, 309. ToRREY, J. G. (1959). Experimental modification of development in the root. Cell, Organism and Mtheu (Ed. by D. Rudnick), pp WiTKUS, E. R. & BERGER, C. A. (1950). Induced vascular differentiation. Bidl. Torrey bot. Club, 77, 301.

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