AN AGEING PROGESS IN EXGISED ROOTS OF GROUNDSEL {SENECIO VULGARIS L.)

Transcription

1 AN AGEING PROGESS IN EXGISED ROOTS OF GROUNDSEL {SENECIO VULGARIS L.) BY J. W. HANNAY* AND D. N. BUTCHERj Department of Botany, University of Manchester [Received 25 January i960) (With 6 figures in the text) SUMMARY The growth and ageing of individual meristems of excised groundsel roots is described. Their behaviour upon repeated subculture is basically similar to that described by other workers for excised tomato roots, although excised groundsel roots require a greater concentration of sucrose for optimal growth than do excised tomato roots. However, growth in low-sugar media can be considerably improved by adding small amounts of 2-naphthoxyacetic acid. When roots grow vigorously in media containing the optimal sucrose concentration, or in low-sugar media supplemented with 2-naphthoxyacetic acid, they show signs of ageing after a few passages and usually stop growing after about 10 weeks. Roots in low-sugar media grow less vigorously but survive longer, i-naphthoxyacetic acid is a very effective 'anti-ageing' substance in that it enhances survival of roots in high-sugar media, or in media supplemented with 2-naphthoxyacetic acid. The ageing process appears to be under the control of a hormonal factor which is not P-indolylacetic acid. INTRODUCTION The term ageing has been used as a convenient description of a process in excised roots which is characterized by a particular pattern of growth. When roots were grown for weekly periods in standard medium, after which the 10 mm apical portions of the roots were excised and transferred to fresh flasks of standard medium, then, initially, the roots grew well and increased in vigour for 2 to 3 weeks. Following this they usually maintained a high rate of growth for several weeks but eventually there was a marked decline in vigour and finally the main apices ceased to elongate, became brown and swollen and sank to the bottom of the flasks. If an individual excised root was allowed to grow for several weeks in a large volume of medium without excising the main apex, it was found that lateral roots continued to grow after the main apex had become brown and ceased to elongate and that secondary laterals might continue to grow after the primary laterals had died. Street et al. (19S2-55) first described such a sequence of events in excised tomato roots and they made considerable progress in elucidating some of the factors involved. It was concluded that the ageing was not due to changes occurring in the external medium ('staling') but that it was probably caused by the accumulation of a hormone * Now at Department of Plant Physiology, Imperial College of Science and Technology, London. t Now at Department of Botany, University College of Swansea.

2 IO J. W. HANNAY AND D. N. BUTCHER within the root. It was suggested (Street, 1954) that this hormone was produced in supra-optimal amounts by vigorously growing roots but that the hormone was itself essential for growth. Skinner (1953) showed that excised groundsel roots could be cultivated in a medium similar to that used for excised tomato roots. However, unlike tomato roots, the excised groundsel roots did not seem to show any signs of ageing. The present investigation was undertaken to compare the behaviour of excised groundsel roots with the previous observations of Street et al. on tomato. Groundsel was chosen not merely because it differed from tomato but also because it seemed to be convenient material for a subsequent investigation of the behaviour of the roots on plants grown in solution culture. EXPERIMENTAL Cultttre technique The clone of excised roots used in this investigation was derived from a single seedling of groundsel {Senecio vulgaris L.). Our particular strain of groundsel was collected in Iceland and is similar to that used by Skinner and Street (1954) and Charles and Street (1959). The general technique for cultivation of the excised roots was similar to that described by Skinner and Street (1954) with the following modifications. The medium used for cultivating the clone was similar to that used by Boll and Street (1951) except that glycine was omitted, manganese was reduced from 1.65 p.p.m. to o.i p.p.m. Mn and 2.5 X 10" g/ml 2-naphthoxyacetic acid was added. In this modified medium growth was more uniform and more vigorous than in that used by Skinner and Street (1954) and by Charles and Street (1959). In maintaining the clone a 'sector passage' was alternated with a 'tip passage'; the inocula for experiments being the 10 mm apical portions of the primary lateral roots of the sector cultures (Street, McGonagle and Lowe, 1951). Each passage is a period of 7 days incubation in the dark at 26 ±1 C. In the tables and figures contained in the text the data quoted are the mean values for ten individual replicates per treatment unless stated otherwise. A root was considered to have died when it had turned brown, sunk to the bottom of the Hask and had not grown more than 10 mm in length during 7 days incubation. The tips of such roots usually had a characteristic hooked appearance and showed no further elongation even if they were subcultured into fresh medium. Sucrose concentration Street, McGonagle and Roberts (1953) showed that the concentration of sucrose m the medium profoundly affected the rate of ageing in excised tomato roots. The data of Table i and Eig. i, which are from the same experiment, show the effects of sucrose concentration upon the growth and survival of excised groundsel roots. At the end of each week (each passage) the 10 mm apical portion of the main axis of the root was excised and transferred to a fresh flask of the appropriate medium. The data of Table i show that greatest linear growth occurred in a medium containing 3% sucrose. However, the survival data of Fig. i clearly show that roots grown in 3 % sucrose aged and died much more rapidly than those grown in lower concentrations of sucrose. Increasing the sucrose concentration beyond 3'^',, produced poorer growth.

3 Ageing in groitndsel roots Thus groundsel roots grew best in a modified White's medium containing the enhanced sugar concentration of 3'^o sucrose instead of the normal 2% sucrose. Under these conditions they showed a similar pattern of growth and ageing to that described by Street et al. for tomato roots. When excised groundsel roots were grown in media containing 2"o sucrose they had a sub-optimal concentration of some essential growth II ; 6 0 A k 0 ti A a sucrose cone. 1 % 2% 3% 4% 6% I Passage number Fig. I. The effect of sucrose concentration upon the survival of excised groundsel roots. factor since they grew more vigorously in 3% sucrose. The factor which was essential for a high growth rate also seemed to be correlated with the ageing phenomenon since roots tended to die after a period of vigorous growth. If growth was depressed by reducing the sucrose concentration below 3% (Table i) then death of the meristem was either Table i. The effect of sucrose concentration upon the growth of excised roots of grou?idsel Passage Passage 2 Sucrose ImA LN LL IMA LN concentration (mm) (mm) (mm) % 2 % 3% 4% 5% III I IO III LL (mm) 16 IOI IMA (mm) Passage 5 Passage 8 LN LL IMA LN (mm) (mm) I " IMA = Mean increase in length of the main axis of the root. LN = Mean number of laterals per root. LL Mean total length of laterals per root. LL (mm) II 18

4 12 J. W. HANNAY AND D. N. BUTCHER C 6 s 120 E E Passage number Fig. 2. The effects of 2-naphthoxyacetic acid (2-NOA) upon the growth and survival of excised groundsel roots.

5 Ageing in groundsel roots 13 delayed or prevented (Fig. i). On the other hand, if growth was retarded by increasing the sucrose concentration beyond the optimal then ageing was usually accelerated. Unpublished data show that, within the concentration range shown m Fig. i, the effects of sucrose are not due to its osmotic influence. For example, roots grown with 2'Jo sucrose plus 2% mannitol grow like roots in 2% sucrose and not like roots in 6% sucrose. 2-naphthoxyacetic acid Charles and Street (1959) have shown that the synthetic growth substance 2-naphthoxyacetic acid (2-NOA) can produce a very marked stimulation of the growth of excised groundsel roots when added to a medium containing 2",, sucrose. An experiment was therefore carried out to test whether the stimulation of growth produced by adding 2-NOA would lead to subsequent ageing of groundsel roots ~ as the stimulation by added sucrose had done. The data of Fig. 2 showed that this was the case. Addition of 3.75 X io~" g/ml or io"'-' g ml 2-NOA to the standard 2']o sucrose medium caused a very considerable increase in lateral development in the early passages (Fig. 2(c). 2% SUCROSE 3 SUCROSE ^ ^ " u I Passage number Passage number Passage number H (-Control Q O2.5X lo-^g./ml. 2NOA added _Q5.0x IO-»g./ml. 2NOA added /^ /\i7.5>' lo-'g./ml. 2NOA added Fig. 3. The effects of the interaction between sucrose and 2-naphthoxyacetic acid (2-NOA) upon the growth and survival of excised groundsel roots. Although only the data for total length of laterals per root have been plotted, the flgures for mean number of laterals per root would give similar curves. This increase in growth was accompanied by a marked increase in the rate of ageing of the roots (Fig. 2(a). Thus either extra sucrose or addition of 2-NOA to the standard medium caused an increase in growth followed by an increase in the rate of ageing. As might be anticipated,

6 J. W. HANNAY AND D. N. BUTCHER 10 Cone. IAA -X Nil -O lo-'ig.lml. -A 10' g.lml. - I0-' g./ml S E 60 - E E E 300 ii o Passage number Fig. 4. The effect of P-indolylacetic acid (IAA) upon the growth and survival of excised groundsel roots.

7 Ageing in groundsel roots 15 the sucrose and 2-NOA were shown to be complementary in their effects upon the growth and ageing process (Fig. 3). High sugar enhanced the effect of a given amount of 2-NOA. These results suggest that the factor which limits growth and ageing in 2% sucrose medium may not be a lack of sugar. The stimulatory effect of 3% sucrose, as against 2% sucrose, could be indirectly due to an increase in the production of a natural hormone. If this is so, then 2-NOA may substitute for this natural hormone. Alternatively, the 2-NOA may promote the uptake, or utilization, of sucrose. Unfortunately, the latter possibility is not very easy to test directly. ^-indolylacetic acid The obvious natural hormone which might be involved is P-indolylacetic acid (IAA), although Street (1954a) has concluded that IAA is not directly involved in the ageing of excised tomato roots. The effect of IAA on the growth and survival of groundsel roots is shown in Fig. 4. The data show that IAA had a different effect on growth and survival of the roots from either increased sucrose or added 2-NOA. Although addition of IAA caused some stimulation of lateral growth, the effect was small compared with that of 2-NOA. Thus 2 X io"' g/ml IAA, which produced the greatest stimulation of lateral growth, inhibited the growth of the main axis considerably but did not increase the rate of ageing of the roots. When the interaction of IAA and sucrose was tested (Fig. 5) it was found that the relative effect of a given concentration of IAA was independent of the sucrose concentration, whereas with 2-NOA the curves for 2 o and 3% sucrose showed a distinct crossover. If it is assumed that increasing the sucrose concentration, or addition of 2-NOA, to 2% sucrose media affect the growth and ageing of the roots in a similar way, then it can be foreseen that the amount of 2-NOA needed to give optimal growth (or the first sign of inhibition) would be less in 3% sucrose media than in 2% sucrose media. Hence a cross-over in the curves as shown in Fig. 5 would be anticipated. In addition it has been postulated that the natural ageing of the roots with increasing passage number results from accumulation of the ageing factor. This would explain why the sensitivity of the roots to added 2-NOA increases from Passage 2 to Passage 3, e.g. in Passage 2 and 2% sucrose, ^' g/ml 2-NOA produced the best growth of laterals whereas in Passage 3 this quantity of 2-NOA was supra-optimal. The effect of adding sucrose could not be simulated by adding IAA. It appeared, therefore, that IAA was not directly responsible for the growth and ageing behaviour under investigation. I -naphthoxyacetic acid An indication of the nature of the substance responsible for stimulating growth and accelerating death may be found in the fact that its effects could be mitigated by addition of I-naphthoxyacetic acid (i-noa). The close similarity between this 'anti-ageing' substance and the 'ageing' substance 2-NOA suggests that they compete at the same active centre. When I-NO A was added to standard medium the main effect was a drastic suppression of lateral development but at 5.0 X io"" g/ml i-noa, the growth of the main axis was also noticeably reduced (Tables 2 and 3). The interaction between 2-NOA and i-noa B N.P.

8 i6 J. W. HANNAY AND D. N. BUTCHER is illustrated in Table 2. The deleterious effects of 7.5 x io"" g/ml 2-NOA on survival of the roots were completely removed when sufficient i-noa was added. Similarly, with 7.5 X10-' g/ml 2-NOA, growth was less in Passage 2 than in Passage i whereas passage two passage three O ine c 20 A O A-- -- L 0 --k --# 2-NOA series 3 % suerose 2% suerose IAA series 3 % sucrose 2"/ sucrose 1 t 1 \ 1 * I 1 \ \ \ \ \ \ \l M 60 to 20.og IAA cone (g.lml.)v 2-NOA cone. (g.lml. 10') 10 " ' 25 IO-'» ' ' E "^ 400 ^ Log IAA cone (g Iml ) Q'^'?-hJOA eonc (g /ml I Fig. 5. A comparison of the effects of 2-naphthoxyacetic acid (2-NOA) and 3-indolylacetic acid (IAA) upon the growth and sun'i\'al of excised groundsel roots. (Data for each growth substance from a separate experiment.) this was reversed by addition of i-noa to the normal trend, in which growth improved in the second passage. Likewise the deleterious effects of added i-noa, particularly on lateral development, were alleviated by adding 2-NOA.

9 Ageing in groundsel roots 17 It appeared that i-noa and 2-NOA had opposite effects upon the ageing behaviour of excised groundsel roots, and also upon their growth. Further confirmation of the different effects of 2-NOA and IAA upon growth were obtained by comparing the interaction between i-noa and 2-NOA (Table 2) with the interaction between i-noa and IAA (Table 3). It should be noted that the depression in growth of the main axis produced by 2 x io"" g ml IAA was accentuated, rather than alleviated, by addition of I-NOA. Table 2. The effects of i-naphthoxyacetic acid and 2-naphthoxyacetic acid on the growth and survival of excised groundsel roots in standard medium i2% sucrose) Concentration Concentration of 2-naphthoxyacetic acid (g/ml io") ofi-naphth- Passage o oxyacetic acid number Number* IMAt LNj LLf Number IMA LN LL Number IMA LN LL (g/mly 10') surviving (mm) (mm) surviving (mm) (mm)surviving(mm) (mm) o I I II II O I o o * Number of roots which had grown more than 10 mm and had not gone brown and sunk to the bottom of the flask during the 7 day incubation period. t Legend as in Table i. The apparent 'anti-ageing' activity of i-noa, was next tested in media containing various concentrations of sucrose. A range of concentrations of i-noa was tested against 2.0, 3.5 and 4.5% sucrose. The results are shown in Fig. 6. Fig. 6 (a) shows survival of roots in the various sugar media without any added i-noa. Roots died fairly rapidly in 4.5% sucrose but survived well in 2% sucrose. Fig. 6(b) shows that the number of roots surviving at the end of Passage 8 increased with increasing concentration of i-noa in media containing either 3.5% or 4.5% sucrose. Death of the roots could be prevented completely by addition of 3.0' id~' g/ml i-noa. In this respect, i-noa is therefore an effective 'anti-ageing' substance. Table 3. The interaction between i-naphthoxyacetic and ^-indohlacetic acid upon the growth of excised groundsel roots in standard medium (2^0 sucrose) Concentration Concentration of P-indolylacetic acid (g/ml) of i-naphth- Passage o 2> io~"' 2 ixyace:tic acid number Number IMA LN LL Number IMA LN LL Number IMA (g/ml Xio') surviving (mm) (mm) surviving (mm) (mm) surviving (mm) LN I no r I IOI I LL (mm) DISCUSSION These data for groundsel roots can be interpreted using the hypothesis of Street (1954a) which suggested that ageing is due to the accumulation of a hormone which, whilst essential for growth, is produced in supraoptimal amounts by vigorously growing roots. The evidence suggests that IAA is not directly involved. However, the effects of the ageing hormone in groundsel roots can be simulated by 2-NOA and counteracted by

10 i8 J. W. HANNAY AND D. N. BUTCHER I-NOA. Hansen (1954) also found that 2-NOA and i-noa had opposite effects upon the growth of seedling wheat roots. i-noa acted as an anti-auxin by increasing cell elongation and the term 'root auxin' was proposed for such substances. 2-NOA acted like a 'shoot auxin' and depressed both cell elongation and cell division in the seedling roots. However, i-noa and its derivatives differed from the other root auxins because they did not increase the positive geotropic reaction. Roberts (19S9) reported that (a) >-i Passage number (b) Ke/ as above E Z ' 3 10 Log concentration of I NOA (g./ml. : 10*) 30 Fig. 6. (a) The effect of sucrose concentration upon the survival of excised groundsel roots, (b) The effects of the interaction betvi'een i-naphthoxyacetic acid (i-noa) and sucrose upon the survival of excised groundsel roots. (Data for (a) and (b) from the same experiment.) I-NOA actually reversed the geotropic response of seedling rice roots; several other substances were tested but only i-noa produced this effect. Taken in conjunction with Street's (1955) findings concerning the nature of anti-ageing substances for excised tomato roots these results seem to indicate that i-noa, and closely related substances, may have rather special effects upon root growth. Such substances may have little effect on shoot growth since they are virtually inactive in most of the standard methods of auxin assay (Fawcett et al., 1953).

11 Ageing in groundsel roots 19 There is an important reservation about the action of i-noa as an anti-ageing substance for excised groundsel roots. In Table 2 the data shov^'ed that the ageing effects induced by 2-NOA were counteracted by i-noa. Not only was survival enhanced but growth of the main axis was also improved. When ageing was induced by high sugar, however, i-noa would increase survival but would not improve growth. Table 4. The effect of sucrose concentration upon the growth and survival of excised groundsel roots and its interaction zvith l-naphthoxyacetic acid (l-noa) 2. o% 3-5% 4-5% 4-5"o 3< 4-5"o 3 ' Treatment Sucrose Sucrose Sucrose Sucrose plus IO"' g/ml I-NOA Sucrose plus io-'g/ml I-NOA Passage Number IMA surviving (mm) IO I LN LL Numbe r Passage 2 IMA LN (mm) surviving (mm) IO Passage LL : Xumber IMA (mm) surviving (mm) LN LL (mm) The data of Table 4 were taken from the same experiment as Fig. 6 and it can be seen that the depressing effect of 4.5% sucrose, as compared with 3.5% sucrose, was actually increased rather than alleviated by i-noa. This must mean that death of roots in high sugar media is not completely similar to that occurring in 2-NOA media. It may be that high sugar produces other deleterious side effects as well as enhancing the production of ageing substance. Alternatively, it may mean that i-noa will counteract the effect of 2-NOA because their chemical similarity allows them to compete at the same active site; but that the actual ageing substance whose production would be affected by high sugar is not acting at the same site. Other natural growth substances such as gibberellic acid and kinetic could be concerned in the ageing process although preliminary results (Wren and Hannay, unpublished) suggest that these particular substances are not directly involved. An attempt is being made to extract growth substances from excised groundsel roots in a search for the hypothetical ageing hormone. Britton, Housley and Bentley (1956) have shown that excised tomato roots contain several growth substances, some of which were neither indolylacetic acid nor indolylacetonitrile. Preliminary experiments with excised groundsel roots have given similar results. REFERENCES BOLL, W. G. & STREET, H. E. (1951). Studies on the growth of excised roots. I. The stimulatory effect of molybdenum and copper on the growth of excised tomato roots. Neiv PhytoL, 50, 52. BRITTON, G., HOUSLEY, S. & BENTLEY, J. A. (1956). Studies in plant growth hormones. V. Chromatography of hormones in excised roots and intact roots of tomato seedlings. J. Exp. Bot., 7, 239. CHARLES, H. P. & STREET, H. E. (1959). Studies on the growth of excised roots. VI. The effect of certain amino acids and auxins on the growth of excised groundsel roots. New PhyloL, 58, 75. FAWCETT, C. H., OSBORNE, D. J., WAIN, R. L. & WALKER, R. D. (1953). Studies on plant growth regulating substances. VI. Side-chain structure in relation to growth-regulating activity in the aryloxyalkylcarboxylic acids. Ann. Appl. Biol., 40, 232. HANSEN, B. A. M. (1954). A physiological classification of 'shoot auxins' and 'root auxins'. II. Bot. Nottser., 318. ROBERTS, E. H. (1959). Geotropic and morphological alterations in rice seedlings caused by plant growth regulators. Nature, Lond., 183, SKINNER, J. C. (1953). Genetical and physiological studies of the behaviour of excised root cultures of groundsel Senecio vulgaris L. Ph.D. Thesis, Manchester. SKINNER, J. C. & STREET, H E. (1954). Studies on the growth of excised roots. II. Obser\ ations on the growth of excised groundsel roots. Neiv PhytoL, 53, 44. STREET, H. E. (1953). Factors controlling meristematic activity in excised roots. III. Light as a factor in the 'location effect' noted with roots of Lycopersicum esculantum Mill. Physiol. Plant., 6,

12 20 J. W. HANNAY AND D. N. BUTCHER STREET, H. E. (!y54 (). Factors controlling meristematic activity in excised roots. V. EfFects of 3-indolylacctic acid P-indolylacutonitrilL- and a-(i-naphthylniethylsulphide)-propionic acid on the growth and survival of roots of Lycopersicum esculenliim Mill. Physiol. Plant., 7, 212. STREET, H. E. (19546). Effect of alpha-(i-naphthymethylsulphide)-propionic acid on the growth of excised tomato roots. Nature, Lond., 173, 253. STREET, H. E. (1955). Factors controlling meristematic activity in roots. VI. Effects of various 'antiauxins' on the growth and survi\al of excised roots of Lycopersicum escidentum Mill. Physiol. Plant., 8, 48. STREET, H. E. & MCGON.\CLE, M. P. (1953). Factors controlling meristematic activity in excised roots. IV. Habiluation of the main axis meristem of excised tomato roots to repeated subculture. Physiol. Plant., 6, 707. STREET, H. E., MCGONACLE, M. P. & LOWE, J. S. (1951). Observations on the 'staling' of White's medium by excised tomato roots. Physiol. Plant., 4, 592. STREET, H. E., MCGON.^CLE, M. P. & ROBERTS, E. H. (1953). Factors controlling meristematic activity in excised roots. 11. Experiments involving repeated subculture of the main axis meristem of roots of Lycopersicum esculentum Mill, and Lycopersicum pimpinellifolium DunaL Fhysiol. Plant., 6, i. STREET, H. E. & ROBERTS, E. H. (1952). Factors controlling meristematic activity in excised roots. L Experiments showing the operation of internal factors. Physiot. Plant., 5, 498.

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