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Some effects of a fulvic acid component of soil organic matter on the growth of cultured excised tomato roots
Sod Bml Bmchrm. Vol 8. pp. 511 to 517. Pergmon
Press 1976. Printrd
I” Great Britain
SOME EFFECTS OF A FULVIC ACID COMPONENT OF SOIL ORGANIC MATTER ON THE GROWTH OF CULTURED EXCISED TOMATO ROOTS D. J. The Macaulay
Institute
for Soil Research, (Accepted
LINEHAN
Craigiebuckler, 15 March
Aberdeen,
AB9 2QJ, Scotland
1976)
Summary-The growth rate of excised tomato roots in culture depends on the age of the root. The rate increases for 10 days and then slows down. The response of such roots to the presence of fulvic acid (FA) in the nutrient medium also depends on the age of the root. Initially the presence of FA has little effect on the growth rate but after 6-7 days the rate exceeds that of controls and continues at this rate for a further 14 days. An analysis of this change in the growth pattern of cultured tomato roots can more easily be made if. after 7 days’ growth, the primary root tip is excised into fresh medium and culture continued for a further 7 days. In this system, main axis extension and the development of laterals as well as the fresh weight of the root is increased by the presence of FA in the medium. Fresh weight is increased despite a depression in the number of cells in the primary root because cell expansion is increased by FA to an extent which more than compensates for the depressed cell numbers. Increased cell expansion is accompanied by an increase in cellular protein. The iron nutrition of the roots is depressed by the presence of FA but this does not appear to be responsible for the enhanced growth. The effects of FA in the nutrient medium have been shown to bersist even after the transfer of the roots to FA free media. INTRODUCTION
Humified organic matter or humus. because of its colloidal nature, has a major influence on the physical properties of soils and consequently on the growth of plants in soils. The various components of humus also possess chemically reactive groups which may influence plant growth directly. To investigate this latter possibility whilst avoiding growth responses resulting from the physical effects of humic substances on soil structure many workers have extracted organic matter from soil and examined its effects on plants growing in solution culture (Kononova, 1966). This approach simplifies the problem considerably but, because of the complex nature of humus, such extracts may influence growth in a number of ways either directly or indirectly. Indirect effects would include the supply and regulation of plant nutrients in a manner analogous to the way in which synthetic ion exchangers operate. Direct effects could result from the uptake of components of the humic material which might alter the distribution of nutrients within the plant or might exert a direct influence on the growth regulation of the plant (Schnitzer and Kahn, 1972). The structural and biochemical complexity of higher plants makes distinction between these possibilities difficult. The problem can be further simplified by the use of plant tissue or organ cultures which are structurally simpler and because of this have simpler growth regulation systems. Various higher plant tissues and organs can be cultured on simple chemically defined media and have been used in investigations of natural and synthetic growth regulating substances (Mitchell and Livingston, 1968). The present paper describes the effect of some extracts OThe
Macaulay
Institute
for Soil Research.
1976. 511
of soil organic matter on the growth of cultured tomato roots whose nutrition and physiology have already been thoroughly investigated (Street, 1966). MATERIALS AND METHODS Preparation matter
of the filoic
acid fraction
of soil
organic
Organic matter was extracted from soils of four Scottish soil series (Glentworth and Muir, 1963). These were: Countesswells series, a well-drained soil derived from granite and granitic gneiss; Insch series, a well-drained soil formed on basic-igneous rocks; Drumlassie series, a poorly-drained sandy loam derived from granite and granitic gneiss; Boyndie series, a very freely-drained soil whose parent material was fluvio-glacial sand. The National grid references of the sites from which these soils were collected are NJ905045; NJ785255; NJ645059 and NJ147658, respectively. Air-dried soil was extracted with 0.5 M NaOH and the humic acid precipitated by acidification to pH 1.0 with 50”, (v/v) HCl (Vaughan, 1969). The acid-soluble fulvic acid (FA) material was then fractionated by Forsyth’s procedure (1947). FA was adsorbed onto animal charcoal and its components eluted successively with water, 90”;, (v/v) aqueous acetone. water. and lastly 0.2~ NaOH. The fraction used in this investigation was that eluted with aqueous acetone and designated fraction B by Forsyth (1947). The solvent was removed by thin film evaporation and the organic material freeze-dried. In one experiment FA was adsorbed onto charcoal, washed with water and eluted with 0.2 M NaOH. The resulting alkaline solution was neutralised with HCl and the NaCl removed by membrane ultrafiltration (Baker, 1970) using an Amicon UM2 membrane having a molecular weight exclusion limit of 1000.
512
D. J.
LINEHAN
Chemical analysis of the Insch FA eluted from charcoal with 90”” (v/v) aqueous acetone showed 4_5.64j0C. 3.3”, H. 51.1”,, 0, X.X and 6.0m-equivalent of COOH and weakly acidic OH respectively per g of dry ash free FA. Its number average molecular weight was 760. Its ash content was 0.35”, and it was completely soluble in water. This component appears to be relatively abundant in agricultural soils occurring in amounts of from I.&IO.0 g. kg- ’ soil in those soils so far investigated. Excised
root
cultwr
The aseptic nutrient medium found to be optimal for growth of cultured roots of the tomato variety Moneymaker was modified from that of Street’s, group (Street and Henshaw, 1966) and was of the fol1.3 mM; lowing composition: Ca(N03)24H,0. MgS0,7H20, 1.2 mM; KCl, 0.9 mM: KNO,. 0.8 mrvr: NaH,PO,H,O, 140p~ NaBO,, 30~~: KI. 4.5 PM; 0.2 PM: MnS0,4H,O, 1.5 /lM; ZnS0,7Hz0, CuS0,5HI0. 40 nM; MOO,. 7 nM. To these nutrient salts were added ferric citrate, 20~~: nicotinic acid. 4 PM; thiamine. 0.3 ,UM: pyridoxine, 0.5 L!M: and sucrose, 58 mM. This was the standard medium to which FA was added where appropriate. After adjustment of their pH to 5.0 with HCl or KOH the medium was sterilized by filtration through membrane filters of 0.22 pm pore size, 20 ml portions being filtered into 90mm dia polystyrene Petri dishes or 200ml into I 1 Roux flasks. Seeds of tomato (Ly.xywrsicw CSC.II/L’~Z~W~~ Mill cultivar Moneymaker) were surface sterilised by immersion for 3 min in 0.5”,, (v/v) peracetic acid. and after washing with sterile distilled water were transferred to Petri dishes containing 20 ml of I”,, (w:‘v) wateragar where they were allowed to germinate for 4 days at 25°C in the dark. Seeds having radicles about 20 mm long were selected and the apical IO mm excised. One such root apex was transferred aseptically into each Petri dish or flask of medium and incubated at 25 C in the dark for 7 or more days, In those experiments involving subculturing of the root apex. the apical 10mm was again excised from each root and transferred aseptically to fresh media to be incubated for a further 7 days. These two culture periods are referred to as first and second passages (Street and Henshaw. 1966). Measurement
of‘ grmvtk
Total linear growth of the roots was measured in mm and the number of laterals counted at the end of each culture period. The total number of cells in the main axis during the culture period was determined by excising the lateral roots as close as possible to the main axis. weighing the main axis tissue, macerating it in 5”,, chromic acid for 24 h and counting the number of cells using a haemocytometer. The average weight of each cell was calculated from the fresh weight of root tissue and the number of cells. Protein was extracted by grinding the root tissue in pH 5.0 citrate phosphate buffer and precipitating the protein with lo”,, (w/v) trichloracetic acid at 1 C for 24 h and was determined using the automated Folin method of Bartley and Poulik (1966) with bovine serum albumin as a standard.
RESULTS
AND DISCUSSION
The rate of increase in the fresh weight of a IOmm root tip. excised from a germinating tomato seed into standard nutrient medium, increased progressively to a maximum rate 10 days after excision. The rate then started to decrease so that after a further 10 days the rate had fallen to about one tenth of the maximum (Fig. 1). The presence of 50mg.l-’ of Insch FA in the nutrient medium produced a considerable change in the growth pattern of the cultured tomato root (Fig. 1). During the first 6 days of culture the presence of the organic matter had no significant effect on the rate of fresh weight increase. At 8 days the rate exceeded that of controls in standard nutrient media. This maximum rate was maintained for several days and only after about 14 days’ culture did it begin to decreas?. Prolonged culture results in the development of such a complex branched structure that detailed analysis of growth becomes difficult. Extended culture periods also produce problems of nutrient depletion and the build-up of toxic concentrations of metabolic products. Street (1966) developed a procedure largely overcoming these problems in‘which the primary root tip is excised after 7 days’ culture and aseptically transferred to fresh nutrient medium. It was shown that changes in the growth of the primary root depend on the activity of the primary meristem and that these changes are qualitatively similar whether culture is continued over an extended period without interference or the root apex excised and transferred to fresh media at 7 day intervals (Street, 1967). The growth patterns of roots cultured for three 7 day periods in standard media or media containing 50 mg.l-’ Insch FA can be seen (Fig. 2) to be broadly similar to those of roots cultured without re-excision of the root apex during a IO-day period (Fig. 1). The major differences were firstly that after 7 days the absolute growth rates were higher in the non-subcultured roots where the growth of laterals was making a major contribution to the increase in fresh weight. Secondly in media containing FA the growth rate of non-subcultured roots (Fig. 1) tended to slow down after I4 days’ growth whereas, when the root apex
2or
oPyFo
18 7 9 r
I.
t I 16
\
14 :
0
2
4
6
8
Time,
IO
12
14
16
18
20
22
days
Fig. I. The growth rate of excised tomato roots cultured in standard medium (solid symbols) or medium containing fulvic acid (open symbols). Values are the means 50 mg.l-’ of 10 replicates.
Fulvic
513
acid and root growth
was transferred
0
2
4
6
8 Time,
IO
12 14
to fresh media, growth through both second and
16 18 20
rate
Table 1 shows various roots cultured for two
days
Fig. 2. The growth rate of excised tomato roots cultured for three successive 7 day passages in standard medium (solid symbols) or medium containing 50 mg,l-r fulvic acid (open symbols). Values are the means of 20 replicates.
X%&l0
indices
of growth
29 k 2 27 t 3
31 ?L1 39 t 2
1.4t 0.1 2.7LO.1
1m+12 210t 1b
37 t 4 61i6
35 t 2 w+3
1.4 e.0.1 3.1to.1
196+ 12 212t 15
46+5 48+_6
bOt3 48i.3
1.8to.1 3.2t 0.2
195+ 9 229L 13
46tb 63 + 5
at.4
1.9 t 0.1
76 k 4
4.3~0.2
166*10 198t lb
b3 t 6
39 t 2 58 f 4
1.5to.1 3.7f 0.2
l?bf 7
66 t
7
Table 2. The growth of cultured tomato roots during two successive passages in nutrient media containing 25 mg.l-’ fulvic acid from four Scottish agricultural soils. Values are the means of 30 replicates f S.E. of the mean Passage
Main axis length
Number of
Fresh weight
number
Cm)
laterals
(w)
Soil series
Imch
Countesaxells
DrumlClSSie
BOpiie
at
of tomato
7 day passages in nutrient media containing various concentrations of lnsch FA. In both first and second passage roots. main axis extension. fresh weights, dry weights and the numbers of laterals were stimulated by concentrations of FA
Table 1. Growth of cultured excised tomato roots during two successive 7 day passages in media containing various concentrations of Insch series soil fulvic acid. Values are means of 20 replicates k SE. of the mean
None
continued
third culture passages. This agrees with the observations of Street (1967) who showed that various growth stimulating hormones were more effective in maintaining rapid growth with subcultured roots than with those maintained in continuous culture. Street suggested that excision resulted in the removal of the major source of a growth inhibiting hormone synthesized in the mature part of the root. Thus the adoption of this culture system should provide a more realistic indication of the effectiveness of any substance in promoting the growth of cultured tomato roots. Consequently it was adopted in further investigations of the growth promoting properties of FA. a high
1
202L.O
36 Jo3
21 t 2
2
203 + 12
3a t 4
3a * 4
1
17&k
46 t 5
23 k 2
2
190 +ll
59 L 4
St4
1
liot
8
37 + 4
21 t 3
2
212 * 10
56 + 3
42*5
1
170* 12
35 L 3
23 t 3
2
239 k 13
56 L 5
66 +_4
1
177%
44t4
28 f. 4
2
277 i. 14
65 L 6
66 t 7
6
9
514
D. J.
LINEHAN
Table 3. Growth of cultured tomato roots during two successive passages in media containing 25 mg.l-’ Insch series soil fulvic acid prepared with or without acetone elution, or an equivalent amount of an acetone eluate of charcoal. Values are the means of 20 replicates * SE. of the mean
157f a
24t 3
13 t 2
1ui
23 + 4
16 t3
9
1
194+ 12
48t5
33 + 3
2
2l9tlO
63 t 4
48+5
193k 13
52 t 4
28 t 4
211+ 9
59 t 4
45 + 6
165t 11
27 t 3
15 +.3
a
25 t 4
16+ 3
150~
with greater stimulation in the up to SOmg.l-‘, second passage. FA preparations from four different Scottish agricultural soils had similar effects on growth (Table 2). The Forsyth (1947) procedure for the fractionation of FA involving the use of acetone, whilst providing material of relatively homogenous chemical composition, is open to the criticism that compounds having growth regulatory properties might be produced by condensation of acetone molecules. It has indeed been shown that under certain conditions such reactions occur and produce substances active in two different gibberellin bio-assays (Briggs, 1966; Mitchell et al., 1969). Table 3 shows the response of cultured tomato roots to an FA prepared without the use of acetone, to a standard acetone eluate of the same FA and to an acetone eluate of charcoal not having FA adsorbed on it. The response of the tomato roots to both FA preparations is similar whereas the non-ful-
vie acetone eluate produces growth not substantially different from the roots in standard media. Because acetone does not appear to produce substances active in influencing the growth of cultured tomato roots and because of the chemical uniformity of the acetone eluate of fulvic acid the acetone eluate has been used in the further investigation of the response of cultured tomato roots to FA. Since development of both main axis and laterals contribute to fresh and dry weight, increases in weight can only be regarded as a crude integral of root growth. The effect of FA on the growth of the primary root was therefore examined in more detail by counting cells of primary roots from which laterals had been removed (Table 4 and Figs. 3 and 4). The major effect of the ageing of the primary meristem of roots cultured in standard media, shown by a comparison of first and second passage roots, was an increase in the numbers of cells in the main axis but a substan-
30
0
25 Fulwc
Fulvic
acid concentrotlon,
mg.16
Fig. 3. The effect of the addition of various concentrations of fulvic acid to standard nutrient medium on the numbers of cells in the main axes of first passage (solid symbols) and second passage (open symbols) cultured tomato roots. Values are the means of 20 replicates & SE.
50
75
acid concentration,
100 IT&~'
Fig. 4. The effect of the addition of various concentrations of fulvic acid to standard nutrient medium on the fresh weights of cells of the main axes of first passage (solid symbols) and second passage (open symbols) cultured tomato roots. Values are the means of 20 replicates k S.E.
Fulvic
515
acid and root growth
Table 4. Comparison of numbers and fresh weight of cells in main axes of first and second passage roots cultured in the absence and presence of 25 mg.l-’ Insch series soil fulvic acid. Values are the means of 20 replicates k S.E. of the mean
standard
mdia
Fultio mAI
17 t 2
+ 0.11
1
1.21
2
2.52 + 0.20
1
1.03 + 0.08
15 + 2
2
1.51 t 0.16
jot.2
7tl
tial reduction in their size, indicated by their fresh weight (Table 4). In contrast in roots cultured in the presence of 25 mg’l-’ FA cells of second passage roots were substantially heavier than those of first passage roots. The effects, on cell numbers and size, of FA at various concentrations up to lOOmg.l-’ are shown in Figs. 3 and 4. All concentrations of FA resulted in a depression in the number of cells in the main axes of both first and second passage roots (Fig. 3) whilst cell size was depressed in first passage roots but stimulated in second passage roots with maximal stimulation at an FA concentration of 25 mg.l-‘. Since the increase in primary root growth, produced by the presence of FA, depends on enhanced cell expansion it seemed possible that this resulted from an increased uptake of water without any concomitant increase in metabolic activity. This was shown not to be the case since the total protein content of cultured roots increased with increasing FA concentration especially during the second culture passage (Fig. 5). The maximum protein content of the roots occurred at an FA concentration of 25 mg’l-’ which is identical with that producing maximum cell size (Fig. 4). Thus increased cell size is accompanied by a parallel increase in protein. Clearly the presence of FA, in a nutrient medium previously established as being optimal for the growth
I
0
25 Fulvic
50 acid
concentration,
75
100 mg.l-’
Fig. 5. The effect of the addition of various concentrations of fulvic acid to standard nutrient medium on the protein content of first passage (solid symbols) and second passage (open symbols) cultured tomato roots. Values are the means of 20 replicates &SE.
0
50
25 Fulvic
acid
75
concentration,
100 mg.l-’
Fig. 6. The effect of the addition of various concentrations of fulvic acid to standard nutrient medium on the iron content of first passage (solid symbols) and second passage (open symbols) cultured tomato roots. Values are the means of 20 replicates + S.E.
of cultured tomato roots, has a major influence on the growth of these roots by affecting cell division, cell expansion and the development of lateral meristerns. Some experiments have been carried out in order to distinguish the mode of action of FA on the growth of cultured tomato roots, that is, to establish whether the growth changes were a nutritional response or depended on a direct effect on the growth regulation of the root. The fact that the responses of the cultured roots were obtained in nutrient medium optimum for growth argues against but does not preclude the possibility of a nutritional effect. My investigations have, however, shown that the uptake of the cations Ca, Mg, Zn, Mn and Fe, which it might be expected could be complexed by FA, is unaffected by the presence of FA in the nutrient medium except in the case of iron. Iron uptake was shown to be progressively depressed by increasing concentrations of FA (Fig. 6). Consequently it seemed possible that the enhanced growth of cultured tomato roots might result from depressed iron uptake. However, this does not appear to be the case since, in an experiment in which the concentration of iron in the nutrient medium was reduced from the 20 pM concentration of the standard nutrient medium to 0.4pM, growth was depressed concomitantly with depressed iron nutrition (Table 5) indicating that, as had been supposed, 20~~ ferric iron was optimal for growth. The contrast between the effects of fulvic acid on the growth of cultured tomato roots and that of depressed iron nutrition is very clear, thus bringing us nearer to the conclusion that FA may exert its influence on growth through a direct effect on the growth regulation of the root. Further evidence for such a direct effect of fulvic acid on growth regulation was obtained from an experiment in which roots were
516
D. J. LINEHAN Table 5. Growth and iron content of cultured tomato roots after two successive 7 day passages in media containing ferric citrate at five different concentrations. Values are the means of 20 replicates k SE. of the mean
cultured 7 days either in standard nutrient media or media containing 15 mg.l-’ Insch FA. After 7 days culture, 10 mm of each root tip was excised and transferred to fresh media in the usual way, except that root tips from standard media were transferred either to fresh standard media or to media containing FA and similarly those from FA into fresh FA containing media or to standard media (Table 6). The presence of FA during both first and second culture passages is necessary for maximum growth since root tips transferred from media containing FA to fresh FA media show substantially greater growth than those transferred from FA media to standard media (Table 6). However the latter roots have more laterals and higher fresh weights than those transferred from standard medium to fresh standard medium. Thus the composition of the medium during the first 7 day passage has an important effect on the subsequent growth of the root tip despite the fact that only 10 mm of root, weighing less than 1.0 mg. is transferred. Such a response seems to lend support to the hypothesis that FA influences growth through a growth regulatory effect rather than through a direct nutritional effect. A nutritional response would be
expected to disappear very rapidly on changing nutrient conditions, whilst a hormonal effect might be longer lived. Street (1967) has suggested that, during the growth of cultured excised tomato roots, growth regulating substances or their precursors are synthesised in the older parts of the roots and translocated to the apical meristem, tending to reach concentrations inducing a reduction of meristematic activity. The presence of any substance in the culture medium which altered the synthesis of these growth substances would thus influence the subsequent growth of the re-excised root tip. Such responses have indeed been demonstrated by Street (1967) using a range of natural and synthetic growth regulators and appear to be similar to those obtained with FA. The FA preparations isolated from agricultural soils in this investigation appear to be. at least superficially. similar to that isolated from a podzol B,, horizon intensively investigated by Schnitzer and his various co-workers (Barton and Schnitzer, 1963: Schnitzer and Kahn, 1977). This latter material was shown to stimulate root initiation by hypocotyl segments of Phusrolus ~~lyaris (Schnitzer and Poapst, 1967: Poapst and Schnitzer. 19701 with maximum
Table 6. Growth of cultured excised tomato roots during two successive 7 day passages. First passage in standard medium or alternatively in medium containing 25 rng.l-’ Insch series soil fulvic acid. Second passage in the same or alternative media. Values are the means of 20 replicates i SE. of the mean
Fulvic acid and root growth activity at a concentration of 3000-6000 mg .I- ‘. This contrasts with maximum activity at 25 mg.l- ’ found in the present investigation. It seems most likely that the difference in response results from the different bio-assays used rather than from intrinsic differences in the FA preparations although this can be ruled out only by a direct comparison of the FA preparations and bio-assay procedures. The low molecular weight of these FA preparations makes complete chemical characterization a possibility in the near future and indeed Schnitzer and his co-workers have already made remarkable progress in this direction (Schnitzer and Kahn, 1972). Although the results presented here show that relatively low concentrations of a major component of soil organic matter can produce considerable changes in the growth process of cultured excised tomato roots clearly it cannot be supposed that similar responses would be obtained with whole tomato plants. It does. however, seem likely that such a physiologically active material would elicit some, perhaps more covert response, even in the whole plant with its more balanced growth regulator systems.
REFERENCES BAKER R. W.
(1970) Ultrafiltration of macromolecular solutions with highNlux membranes. J. appl. Po/ym. Sci. 14. 1197-1114. BARTLEY E. T. and POULIK M. D. (1966) Rapid selection of proper chromatographic systems for protein isolation. In
Technicon Symposium: Automation in Analytical Chemistry. pp. 383-387. Mediad Inc., White Plains. New
York. BARTON D. H. R. and SCHN~TZER M. (1963) A new experimental approach to the humic acid problem. Nurure, Loml. 1%, 2 17-2 18.
517
BRIGGS D. E. (1966) Residues from organic solvents showing gibberellin-like biological activity. Nature. Lontf. 210, 419421. FORSYTH W. G. C. (1947) Studies on the more soluble complexes of soil organic matter-I. Method of fractionation. Biochrm. J. 41, 176-181. GLENTWORTH R. and MUIR J. W. (1963) The soils of the country round Aberdeen, Inverurie and Fraserburgh. hlem. Soil Suw. Gt Br. H.M.S.O. Edinburgh. KONONOVA M. M. (1966) Soil Organic Matter. Pergamon Press, Oxford. MITCHELL J. W. and LIVINGSTON G. A. (1968) Methods qf Studying Plunt Hormones und Growth Reguluting Sub.stance.s. U.S.D.A. Handbook No. 336. U.S.A. Government Printing Office. MITCHELL J. W.. MANDAVA N., PLIMMER J. R., W~RLEY J. F. and DROWNE M. E. (1969) Plant growth regulating properties of some acetone condensation products. Nuture, Land. 223, 1386-1387. POAPST P. A. and SCHNITZER M. (1971) Fulvic acid and adventitious root formation. Soil Biol. Biochrm. 3, 215-219. SCHNI~ZER M. and KHAN S. U. (1972) Humic Substunces in the Enrironmmt. Marcel Dekker Inc.. New York. SCHNITZER M. and POAPST P. A. (1967) Effects of a soil humic compound on root initiation. Nature. Land. 213. 598-599. STREET H. E. (1966) The nutrition and metabolism of plant tissue and organ cultures. In Cells and Tissues in Culture. Methods, Biology und Physiology (E. N. Willmer. Ed.) Vol. 3. pp. 533-624. Academic Press, New York. STREET H. E. (1967) The ageing of root meristems. Symp. Sot. esp. Biol. 21, 517-542. STREET H. E. and HENSHALVG. G. (1966) Introduction and methods employed in plant tissue culture. In Cells und Tissues in Culture. Methods. Biology and Physiology (E. N. Willmer, Ed.) Vol. 3. pp. 459-520, Academic Press. New York. VAUGHAN D. (1969) The stimulation of invertase development in aseptic storage tissue slices by humic acids. Soil Biol. Biochem. 1, 15-28.
Press 1976. Printrd
I” Great Britain
SOME EFFECTS OF A FULVIC ACID COMPONENT OF SOIL ORGANIC MATTER ON THE GROWTH OF CULTURED EXCISED TOMATO ROOTS D. J. The Macaulay
Institute
for Soil Research, (Accepted
LINEHAN
Craigiebuckler, 15 March
Aberdeen,
AB9 2QJ, Scotland
1976)
Summary-The growth rate of excised tomato roots in culture depends on the age of the root. The rate increases for 10 days and then slows down. The response of such roots to the presence of fulvic acid (FA) in the nutrient medium also depends on the age of the root. Initially the presence of FA has little effect on the growth rate but after 6-7 days the rate exceeds that of controls and continues at this rate for a further 14 days. An analysis of this change in the growth pattern of cultured tomato roots can more easily be made if. after 7 days’ growth, the primary root tip is excised into fresh medium and culture continued for a further 7 days. In this system, main axis extension and the development of laterals as well as the fresh weight of the root is increased by the presence of FA in the medium. Fresh weight is increased despite a depression in the number of cells in the primary root because cell expansion is increased by FA to an extent which more than compensates for the depressed cell numbers. Increased cell expansion is accompanied by an increase in cellular protein. The iron nutrition of the roots is depressed by the presence of FA but this does not appear to be responsible for the enhanced growth. The effects of FA in the nutrient medium have been shown to bersist even after the transfer of the roots to FA free media. INTRODUCTION
Humified organic matter or humus. because of its colloidal nature, has a major influence on the physical properties of soils and consequently on the growth of plants in soils. The various components of humus also possess chemically reactive groups which may influence plant growth directly. To investigate this latter possibility whilst avoiding growth responses resulting from the physical effects of humic substances on soil structure many workers have extracted organic matter from soil and examined its effects on plants growing in solution culture (Kononova, 1966). This approach simplifies the problem considerably but, because of the complex nature of humus, such extracts may influence growth in a number of ways either directly or indirectly. Indirect effects would include the supply and regulation of plant nutrients in a manner analogous to the way in which synthetic ion exchangers operate. Direct effects could result from the uptake of components of the humic material which might alter the distribution of nutrients within the plant or might exert a direct influence on the growth regulation of the plant (Schnitzer and Kahn, 1972). The structural and biochemical complexity of higher plants makes distinction between these possibilities difficult. The problem can be further simplified by the use of plant tissue or organ cultures which are structurally simpler and because of this have simpler growth regulation systems. Various higher plant tissues and organs can be cultured on simple chemically defined media and have been used in investigations of natural and synthetic growth regulating substances (Mitchell and Livingston, 1968). The present paper describes the effect of some extracts OThe
Macaulay
Institute
for Soil Research.
1976. 511
of soil organic matter on the growth of cultured tomato roots whose nutrition and physiology have already been thoroughly investigated (Street, 1966). MATERIALS AND METHODS Preparation matter
of the filoic
acid fraction
of soil
organic
Organic matter was extracted from soils of four Scottish soil series (Glentworth and Muir, 1963). These were: Countesswells series, a well-drained soil derived from granite and granitic gneiss; Insch series, a well-drained soil formed on basic-igneous rocks; Drumlassie series, a poorly-drained sandy loam derived from granite and granitic gneiss; Boyndie series, a very freely-drained soil whose parent material was fluvio-glacial sand. The National grid references of the sites from which these soils were collected are NJ905045; NJ785255; NJ645059 and NJ147658, respectively. Air-dried soil was extracted with 0.5 M NaOH and the humic acid precipitated by acidification to pH 1.0 with 50”, (v/v) HCl (Vaughan, 1969). The acid-soluble fulvic acid (FA) material was then fractionated by Forsyth’s procedure (1947). FA was adsorbed onto animal charcoal and its components eluted successively with water, 90”;, (v/v) aqueous acetone. water. and lastly 0.2~ NaOH. The fraction used in this investigation was that eluted with aqueous acetone and designated fraction B by Forsyth (1947). The solvent was removed by thin film evaporation and the organic material freeze-dried. In one experiment FA was adsorbed onto charcoal, washed with water and eluted with 0.2 M NaOH. The resulting alkaline solution was neutralised with HCl and the NaCl removed by membrane ultrafiltration (Baker, 1970) using an Amicon UM2 membrane having a molecular weight exclusion limit of 1000.
512
D. J.
LINEHAN
Chemical analysis of the Insch FA eluted from charcoal with 90”” (v/v) aqueous acetone showed 4_5.64j0C. 3.3”, H. 51.1”,, 0, X.X and 6.0m-equivalent of COOH and weakly acidic OH respectively per g of dry ash free FA. Its number average molecular weight was 760. Its ash content was 0.35”, and it was completely soluble in water. This component appears to be relatively abundant in agricultural soils occurring in amounts of from I.&IO.0 g. kg- ’ soil in those soils so far investigated. Excised
root
cultwr
The aseptic nutrient medium found to be optimal for growth of cultured roots of the tomato variety Moneymaker was modified from that of Street’s, group (Street and Henshaw, 1966) and was of the fol1.3 mM; lowing composition: Ca(N03)24H,0. MgS0,7H20, 1.2 mM; KCl, 0.9 mM: KNO,. 0.8 mrvr: NaH,PO,H,O, 140p~ NaBO,, 30~~: KI. 4.5 PM; 0.2 PM: MnS0,4H,O, 1.5 /lM; ZnS0,7Hz0, CuS0,5HI0. 40 nM; MOO,. 7 nM. To these nutrient salts were added ferric citrate, 20~~: nicotinic acid. 4 PM; thiamine. 0.3 ,UM: pyridoxine, 0.5 L!M: and sucrose, 58 mM. This was the standard medium to which FA was added where appropriate. After adjustment of their pH to 5.0 with HCl or KOH the medium was sterilized by filtration through membrane filters of 0.22 pm pore size, 20 ml portions being filtered into 90mm dia polystyrene Petri dishes or 200ml into I 1 Roux flasks. Seeds of tomato (Ly.xywrsicw CSC.II/L’~Z~W~~ Mill cultivar Moneymaker) were surface sterilised by immersion for 3 min in 0.5”,, (v/v) peracetic acid. and after washing with sterile distilled water were transferred to Petri dishes containing 20 ml of I”,, (w:‘v) wateragar where they were allowed to germinate for 4 days at 25°C in the dark. Seeds having radicles about 20 mm long were selected and the apical IO mm excised. One such root apex was transferred aseptically into each Petri dish or flask of medium and incubated at 25 C in the dark for 7 or more days, In those experiments involving subculturing of the root apex. the apical 10mm was again excised from each root and transferred aseptically to fresh media to be incubated for a further 7 days. These two culture periods are referred to as first and second passages (Street and Henshaw. 1966). Measurement
of‘ grmvtk
Total linear growth of the roots was measured in mm and the number of laterals counted at the end of each culture period. The total number of cells in the main axis during the culture period was determined by excising the lateral roots as close as possible to the main axis. weighing the main axis tissue, macerating it in 5”,, chromic acid for 24 h and counting the number of cells using a haemocytometer. The average weight of each cell was calculated from the fresh weight of root tissue and the number of cells. Protein was extracted by grinding the root tissue in pH 5.0 citrate phosphate buffer and precipitating the protein with lo”,, (w/v) trichloracetic acid at 1 C for 24 h and was determined using the automated Folin method of Bartley and Poulik (1966) with bovine serum albumin as a standard.
RESULTS
AND DISCUSSION
The rate of increase in the fresh weight of a IOmm root tip. excised from a germinating tomato seed into standard nutrient medium, increased progressively to a maximum rate 10 days after excision. The rate then started to decrease so that after a further 10 days the rate had fallen to about one tenth of the maximum (Fig. 1). The presence of 50mg.l-’ of Insch FA in the nutrient medium produced a considerable change in the growth pattern of the cultured tomato root (Fig. 1). During the first 6 days of culture the presence of the organic matter had no significant effect on the rate of fresh weight increase. At 8 days the rate exceeded that of controls in standard nutrient media. This maximum rate was maintained for several days and only after about 14 days’ culture did it begin to decreas?. Prolonged culture results in the development of such a complex branched structure that detailed analysis of growth becomes difficult. Extended culture periods also produce problems of nutrient depletion and the build-up of toxic concentrations of metabolic products. Street (1966) developed a procedure largely overcoming these problems in‘which the primary root tip is excised after 7 days’ culture and aseptically transferred to fresh nutrient medium. It was shown that changes in the growth of the primary root depend on the activity of the primary meristem and that these changes are qualitatively similar whether culture is continued over an extended period without interference or the root apex excised and transferred to fresh media at 7 day intervals (Street, 1967). The growth patterns of roots cultured for three 7 day periods in standard media or media containing 50 mg.l-’ Insch FA can be seen (Fig. 2) to be broadly similar to those of roots cultured without re-excision of the root apex during a IO-day period (Fig. 1). The major differences were firstly that after 7 days the absolute growth rates were higher in the non-subcultured roots where the growth of laterals was making a major contribution to the increase in fresh weight. Secondly in media containing FA the growth rate of non-subcultured roots (Fig. 1) tended to slow down after I4 days’ growth whereas, when the root apex
2or
oPyFo
18 7 9 r
I.
t I 16
\
14 :
0
2
4
6
8
Time,
IO
12
14
16
18
20
22
days
Fig. I. The growth rate of excised tomato roots cultured in standard medium (solid symbols) or medium containing fulvic acid (open symbols). Values are the means 50 mg.l-’ of 10 replicates.
Fulvic
513
acid and root growth
was transferred
0
2
4
6
8 Time,
IO
12 14
to fresh media, growth through both second and
16 18 20
rate
Table 1 shows various roots cultured for two
days
Fig. 2. The growth rate of excised tomato roots cultured for three successive 7 day passages in standard medium (solid symbols) or medium containing 50 mg,l-r fulvic acid (open symbols). Values are the means of 20 replicates.
X%&l0
indices
of growth
29 k 2 27 t 3
31 ?L1 39 t 2
1.4t 0.1 2.7LO.1
1m+12 210t 1b
37 t 4 61i6
35 t 2 w+3
1.4 e.0.1 3.1to.1
196+ 12 212t 15
46+5 48+_6
bOt3 48i.3
1.8to.1 3.2t 0.2
195+ 9 229L 13
46tb 63 + 5
at.4
1.9 t 0.1
76 k 4
4.3~0.2
166*10 198t lb
b3 t 6
39 t 2 58 f 4
1.5to.1 3.7f 0.2
l?bf 7
66 t
7
Table 2. The growth of cultured tomato roots during two successive passages in nutrient media containing 25 mg.l-’ fulvic acid from four Scottish agricultural soils. Values are the means of 30 replicates f S.E. of the mean Passage
Main axis length
Number of
Fresh weight
number
Cm)
laterals
(w)
Soil series
Imch
Countesaxells
DrumlClSSie
BOpiie
at
of tomato
7 day passages in nutrient media containing various concentrations of lnsch FA. In both first and second passage roots. main axis extension. fresh weights, dry weights and the numbers of laterals were stimulated by concentrations of FA
Table 1. Growth of cultured excised tomato roots during two successive 7 day passages in media containing various concentrations of Insch series soil fulvic acid. Values are means of 20 replicates k SE. of the mean
None
continued
third culture passages. This agrees with the observations of Street (1967) who showed that various growth stimulating hormones were more effective in maintaining rapid growth with subcultured roots than with those maintained in continuous culture. Street suggested that excision resulted in the removal of the major source of a growth inhibiting hormone synthesized in the mature part of the root. Thus the adoption of this culture system should provide a more realistic indication of the effectiveness of any substance in promoting the growth of cultured tomato roots. Consequently it was adopted in further investigations of the growth promoting properties of FA. a high
1
202L.O
36 Jo3
21 t 2
2
203 + 12
3a t 4
3a * 4
1
17&k
46 t 5
23 k 2
2
190 +ll
59 L 4
St4
1
liot
8
37 + 4
21 t 3
2
212 * 10
56 + 3
42*5
1
170* 12
35 L 3
23 t 3
2
239 k 13
56 L 5
66 +_4
1
177%
44t4
28 f. 4
2
277 i. 14
65 L 6
66 t 7
6
9
514
D. J.
LINEHAN
Table 3. Growth of cultured tomato roots during two successive passages in media containing 25 mg.l-’ Insch series soil fulvic acid prepared with or without acetone elution, or an equivalent amount of an acetone eluate of charcoal. Values are the means of 20 replicates * SE. of the mean
157f a
24t 3
13 t 2
1ui
23 + 4
16 t3
9
1
194+ 12
48t5
33 + 3
2
2l9tlO
63 t 4
48+5
193k 13
52 t 4
28 t 4
211+ 9
59 t 4
45 + 6
165t 11
27 t 3
15 +.3
a
25 t 4
16+ 3
150~
with greater stimulation in the up to SOmg.l-‘, second passage. FA preparations from four different Scottish agricultural soils had similar effects on growth (Table 2). The Forsyth (1947) procedure for the fractionation of FA involving the use of acetone, whilst providing material of relatively homogenous chemical composition, is open to the criticism that compounds having growth regulatory properties might be produced by condensation of acetone molecules. It has indeed been shown that under certain conditions such reactions occur and produce substances active in two different gibberellin bio-assays (Briggs, 1966; Mitchell et al., 1969). Table 3 shows the response of cultured tomato roots to an FA prepared without the use of acetone, to a standard acetone eluate of the same FA and to an acetone eluate of charcoal not having FA adsorbed on it. The response of the tomato roots to both FA preparations is similar whereas the non-ful-
vie acetone eluate produces growth not substantially different from the roots in standard media. Because acetone does not appear to produce substances active in influencing the growth of cultured tomato roots and because of the chemical uniformity of the acetone eluate of fulvic acid the acetone eluate has been used in the further investigation of the response of cultured tomato roots to FA. Since development of both main axis and laterals contribute to fresh and dry weight, increases in weight can only be regarded as a crude integral of root growth. The effect of FA on the growth of the primary root was therefore examined in more detail by counting cells of primary roots from which laterals had been removed (Table 4 and Figs. 3 and 4). The major effect of the ageing of the primary meristem of roots cultured in standard media, shown by a comparison of first and second passage roots, was an increase in the numbers of cells in the main axis but a substan-
30
0
25 Fulwc
Fulvic
acid concentrotlon,
mg.16
Fig. 3. The effect of the addition of various concentrations of fulvic acid to standard nutrient medium on the numbers of cells in the main axes of first passage (solid symbols) and second passage (open symbols) cultured tomato roots. Values are the means of 20 replicates & SE.
50
75
acid concentration,
100 IT&~'
Fig. 4. The effect of the addition of various concentrations of fulvic acid to standard nutrient medium on the fresh weights of cells of the main axes of first passage (solid symbols) and second passage (open symbols) cultured tomato roots. Values are the means of 20 replicates k S.E.
Fulvic
515
acid and root growth
Table 4. Comparison of numbers and fresh weight of cells in main axes of first and second passage roots cultured in the absence and presence of 25 mg.l-’ Insch series soil fulvic acid. Values are the means of 20 replicates k S.E. of the mean
standard
mdia
Fultio mAI
17 t 2
+ 0.11
1
1.21
2
2.52 + 0.20
1
1.03 + 0.08
15 + 2
2
1.51 t 0.16
jot.2
7tl
tial reduction in their size, indicated by their fresh weight (Table 4). In contrast in roots cultured in the presence of 25 mg’l-’ FA cells of second passage roots were substantially heavier than those of first passage roots. The effects, on cell numbers and size, of FA at various concentrations up to lOOmg.l-’ are shown in Figs. 3 and 4. All concentrations of FA resulted in a depression in the number of cells in the main axes of both first and second passage roots (Fig. 3) whilst cell size was depressed in first passage roots but stimulated in second passage roots with maximal stimulation at an FA concentration of 25 mg.l-‘. Since the increase in primary root growth, produced by the presence of FA, depends on enhanced cell expansion it seemed possible that this resulted from an increased uptake of water without any concomitant increase in metabolic activity. This was shown not to be the case since the total protein content of cultured roots increased with increasing FA concentration especially during the second culture passage (Fig. 5). The maximum protein content of the roots occurred at an FA concentration of 25 mg’l-’ which is identical with that producing maximum cell size (Fig. 4). Thus increased cell size is accompanied by a parallel increase in protein. Clearly the presence of FA, in a nutrient medium previously established as being optimal for the growth
I
0
25 Fulvic
50 acid
concentration,
75
100 mg.l-’
Fig. 5. The effect of the addition of various concentrations of fulvic acid to standard nutrient medium on the protein content of first passage (solid symbols) and second passage (open symbols) cultured tomato roots. Values are the means of 20 replicates &SE.
0
50
25 Fulvic
acid
75
concentration,
100 mg.l-’
Fig. 6. The effect of the addition of various concentrations of fulvic acid to standard nutrient medium on the iron content of first passage (solid symbols) and second passage (open symbols) cultured tomato roots. Values are the means of 20 replicates + S.E.
of cultured tomato roots, has a major influence on the growth of these roots by affecting cell division, cell expansion and the development of lateral meristerns. Some experiments have been carried out in order to distinguish the mode of action of FA on the growth of cultured tomato roots, that is, to establish whether the growth changes were a nutritional response or depended on a direct effect on the growth regulation of the root. The fact that the responses of the cultured roots were obtained in nutrient medium optimum for growth argues against but does not preclude the possibility of a nutritional effect. My investigations have, however, shown that the uptake of the cations Ca, Mg, Zn, Mn and Fe, which it might be expected could be complexed by FA, is unaffected by the presence of FA in the nutrient medium except in the case of iron. Iron uptake was shown to be progressively depressed by increasing concentrations of FA (Fig. 6). Consequently it seemed possible that the enhanced growth of cultured tomato roots might result from depressed iron uptake. However, this does not appear to be the case since, in an experiment in which the concentration of iron in the nutrient medium was reduced from the 20 pM concentration of the standard nutrient medium to 0.4pM, growth was depressed concomitantly with depressed iron nutrition (Table 5) indicating that, as had been supposed, 20~~ ferric iron was optimal for growth. The contrast between the effects of fulvic acid on the growth of cultured tomato roots and that of depressed iron nutrition is very clear, thus bringing us nearer to the conclusion that FA may exert its influence on growth through a direct effect on the growth regulation of the root. Further evidence for such a direct effect of fulvic acid on growth regulation was obtained from an experiment in which roots were
516
D. J. LINEHAN Table 5. Growth and iron content of cultured tomato roots after two successive 7 day passages in media containing ferric citrate at five different concentrations. Values are the means of 20 replicates k SE. of the mean
cultured 7 days either in standard nutrient media or media containing 15 mg.l-’ Insch FA. After 7 days culture, 10 mm of each root tip was excised and transferred to fresh media in the usual way, except that root tips from standard media were transferred either to fresh standard media or to media containing FA and similarly those from FA into fresh FA containing media or to standard media (Table 6). The presence of FA during both first and second culture passages is necessary for maximum growth since root tips transferred from media containing FA to fresh FA media show substantially greater growth than those transferred from FA media to standard media (Table 6). However the latter roots have more laterals and higher fresh weights than those transferred from standard medium to fresh standard medium. Thus the composition of the medium during the first 7 day passage has an important effect on the subsequent growth of the root tip despite the fact that only 10 mm of root, weighing less than 1.0 mg. is transferred. Such a response seems to lend support to the hypothesis that FA influences growth through a growth regulatory effect rather than through a direct nutritional effect. A nutritional response would be
expected to disappear very rapidly on changing nutrient conditions, whilst a hormonal effect might be longer lived. Street (1967) has suggested that, during the growth of cultured excised tomato roots, growth regulating substances or their precursors are synthesised in the older parts of the roots and translocated to the apical meristem, tending to reach concentrations inducing a reduction of meristematic activity. The presence of any substance in the culture medium which altered the synthesis of these growth substances would thus influence the subsequent growth of the re-excised root tip. Such responses have indeed been demonstrated by Street (1967) using a range of natural and synthetic growth regulators and appear to be similar to those obtained with FA. The FA preparations isolated from agricultural soils in this investigation appear to be. at least superficially. similar to that isolated from a podzol B,, horizon intensively investigated by Schnitzer and his various co-workers (Barton and Schnitzer, 1963: Schnitzer and Kahn, 1977). This latter material was shown to stimulate root initiation by hypocotyl segments of Phusrolus ~~lyaris (Schnitzer and Poapst, 1967: Poapst and Schnitzer. 19701 with maximum
Table 6. Growth of cultured excised tomato roots during two successive 7 day passages. First passage in standard medium or alternatively in medium containing 25 rng.l-’ Insch series soil fulvic acid. Second passage in the same or alternative media. Values are the means of 20 replicates i SE. of the mean
Fulvic acid and root growth activity at a concentration of 3000-6000 mg .I- ‘. This contrasts with maximum activity at 25 mg.l- ’ found in the present investigation. It seems most likely that the difference in response results from the different bio-assays used rather than from intrinsic differences in the FA preparations although this can be ruled out only by a direct comparison of the FA preparations and bio-assay procedures. The low molecular weight of these FA preparations makes complete chemical characterization a possibility in the near future and indeed Schnitzer and his co-workers have already made remarkable progress in this direction (Schnitzer and Kahn, 1972). Although the results presented here show that relatively low concentrations of a major component of soil organic matter can produce considerable changes in the growth process of cultured excised tomato roots clearly it cannot be supposed that similar responses would be obtained with whole tomato plants. It does. however, seem likely that such a physiologically active material would elicit some, perhaps more covert response, even in the whole plant with its more balanced growth regulator systems.
REFERENCES BAKER R. W.
(1970) Ultrafiltration of macromolecular solutions with highNlux membranes. J. appl. Po/ym. Sci. 14. 1197-1114. BARTLEY E. T. and POULIK M. D. (1966) Rapid selection of proper chromatographic systems for protein isolation. In
Technicon Symposium: Automation in Analytical Chemistry. pp. 383-387. Mediad Inc., White Plains. New
York. BARTON D. H. R. and SCHN~TZER M. (1963) A new experimental approach to the humic acid problem. Nurure, Loml. 1%, 2 17-2 18.
517
BRIGGS D. E. (1966) Residues from organic solvents showing gibberellin-like biological activity. Nature. Lontf. 210, 419421. FORSYTH W. G. C. (1947) Studies on the more soluble complexes of soil organic matter-I. Method of fractionation. Biochrm. J. 41, 176-181. GLENTWORTH R. and MUIR J. W. (1963) The soils of the country round Aberdeen, Inverurie and Fraserburgh. hlem. Soil Suw. Gt Br. H.M.S.O. Edinburgh. KONONOVA M. M. (1966) Soil Organic Matter. Pergamon Press, Oxford. MITCHELL J. W. and LIVINGSTON G. A. (1968) Methods qf Studying Plunt Hormones und Growth Reguluting Sub.stance.s. U.S.D.A. Handbook No. 336. U.S.A. Government Printing Office. MITCHELL J. W.. MANDAVA N., PLIMMER J. R., W~RLEY J. F. and DROWNE M. E. (1969) Plant growth regulating properties of some acetone condensation products. Nuture, Land. 223, 1386-1387. POAPST P. A. and SCHNITZER M. (1971) Fulvic acid and adventitious root formation. Soil Biol. Biochrm. 3, 215-219. SCHNI~ZER M. and KHAN S. U. (1972) Humic Substunces in the Enrironmmt. Marcel Dekker Inc.. New York. SCHNITZER M. and POAPST P. A. (1967) Effects of a soil humic compound on root initiation. Nature. Land. 213. 598-599. STREET H. E. (1966) The nutrition and metabolism of plant tissue and organ cultures. In Cells and Tissues in Culture. Methods, Biology und Physiology (E. N. Willmer. Ed.) Vol. 3. pp. 533-624. Academic Press, New York. STREET H. E. (1967) The ageing of root meristems. Symp. Sot. esp. Biol. 21, 517-542. STREET H. E. and HENSHALVG. G. (1966) Introduction and methods employed in plant tissue culture. In Cells und Tissues in Culture. Methods. Biology and Physiology (E. N. Willmer, Ed.) Vol. 3. pp. 459-520, Academic Press. New York. VAUGHAN D. (1969) The stimulation of invertase development in aseptic storage tissue slices by humic acids. Soil Biol. Biochem. 1, 15-28.