Pertanika 9(2), 135 -146 (1986)

Effects of Solution, Soil and Sand Cultureson Nodulation and Growth of Phasey Bean

WAN MOHAMAD WAN OTHMAN 1, C.J. ASHER and L.R. HUMPHREYSDepartment of Agriculture,University of Queensland,

St. Lucia, Queensland, 4067, Australia.

Key words: Nitrogen-free nutrient solution; nodulation; nitrogen and dry matter accumulation.

ABSTRAK

Pokok kacang phasey (Macroptilium lathyroides cv... Murray) ditanam di dalam kulturlarutan nutrien tanpa nitrol{en, tanah dan pasir dalam rumah kaca. Pembintilan, tumpukan bahankering dalam bahagian- bahagian pokok dan hasil biji ditentukan. Nitrogen simbiotik yang terbahagidi bahagian-bahagian pokok di peringkat tampang dan mengawan dz'tentukan juga. Di dalam semuamedia kultur bilangan dan saiz bintil meningkat dengan peningkatan umur pokok, tetapi kadarpeningkatan inipada umumnya lebih cepat dalam kultur larutan darzpada kultur-kultur lain. Dalamkultur pasir, berat kering bintd sepokok dan sebintil serta tumbesaran pokok adalah terjejas.Walaupun pemanjangan akar tunjang sentz'asa lebih baik dalam kultur larutan darzpada kulturtanah atau pasir, perkembangan daun dan tumpukan bahan kering dalam akar dan batang diting­katkan oleh kultur larutan hanya di peringkat berbunga dan berbuah sahaja. Hasil bijz' meningkatdengan ketara dalam kultur larutan, satu kesan yang berkaitan dengan pengikatan nz'trogen simbiotikyang tinggi. Semasa pertumbuhan tampang kebanyakan nitrogen tertumpuk dalam daun danbatang, tetapi lenggai adalah sink nz'trogen yang utama semasa peringkat mengawan. Kebaikan dankegunaan kultur larutan dalam kajian perkembangan bz'ntil dan koleksi sampel akar untuk asai-asaipenurunan asetilena dibincangkan.

ABSTRACT

Plants of phasey bean (Macroptilium lathyroides cv. Murray) were grown in nz'trogen-Jteenutrient solution, sod, or sand culture in a naturally-Nt glasshouse. Nodulation, dry matter accu- /'mulation in plant parts, and seed yields were assessed. Partitioning ofsymbz'otic nz'trogen into variousplant parts during vegetative and reproductive growth stages was also determined. In all culturemedia, nodule number and size increased wz'th plant age but the rate of increase was generally greaterin solution than in the other cultures. In sand culture, the dry weight per nodule and per plant, andplant growth were significantly suppressed. A lthough tap root elongation was conszstently better insolution than soil or sand culture, leaf development and dry matter accumulation in roots and stemswere enhanced by solution culture only during flowering and fruiting stage. Seed yields were signifi­cantly increased by solution culture, an effect apparently associated with increased symbiotz'c nitrogenfzxation. During vegetative growth, nz'trogen accumulated largely in the leaves and stems but podswere major sinks of nitrogen during the reproductive growth stage. The benefits and applications ofsolution culture in the study ofnodule development and collection ofroot samples for acetylene reduc­tion assays are dzscussed.

epartment of Agronomy & Horticulture, Universiti Pertanian Malaysia, 43400 UPM Serdang, Selangor, MALAYSIA.

WAN MOHAMAD WAN OTHMAN, C.J. ASHER AND L.R. HUMPHREYS

INTRODUCTION

Sand culture has been utilized in numerousstudies of nodulation and nitrogen fixation ofpasture and grain legumes (Hen~ell 1962 ;Wilson 1972; Andrew 1976; Saito et al., 1980;Jones et al., 1981). However, few comparisonsappear to have been made concerning the valueof solution culture as opposed to other culturemethods for such experiments. It is generallyassumed that nodulation, vegetative growth, andseed yields are unaffected by various plantingmedia under similar environmental and nutri­tional conditions, but there is little experimentalevidence to support or dispute these assump­tions. The present study examines the effects ofsolution, sand and soil culture on nodulation,growth and seed yield of phasey bean, an annualor biennial tropical pasture legume.

The primary objective of this experiment isto investigate whether phasey bean nodulatesfreely in nitrogen-free nutrient solution and toobtain preliminary information on the majorsites of nitrogen accumulation during the vegeta­tive and reproductive growth stages.

MATERIALS AND METHODS

Experimental Design

The experiment consisted of a factorialcombination of three planting media (solution,sand and soil) and six harvests (4, 6, 8, 10, 12and 14 weeks after planting), arranged in acompletely randomized design with four replica­tions. The pots were rerandomized once a monthto minimize any positional effects in the glass­house.

Preparation of Culture Media

Thirty pots (17.5 cm diameter) were eachfilled with 3.23 kg of a 7 : 1 mixture of fine(0.02 - 0.20 mm) and coarse (0.2 - 2.0 mmdiameter) river sand. The sand was previouslywashed under running water for two days, rinsedwith deionized water and then air-dried.Another 30 pots were each filled with 2.63 kg ofa yellow podzolic soil (top 15 cm) taken from the

University of Queensland farm at Mt. Cotton(Field 7) in southeast Queensland. Available soilnitrogen was determined according to themethods described by Bremner and Keeney(1966). and the level (NO 5 - N + NH 4 - N) was12.4 IJ. g g -1. Soil pH (of 5.0) was measured on apH meter using 1 : 5 soil to water ratio. The potscontaining soil and sand were perforated at thebase to allow free drainage. The bulk densities ofsoil and' sand were 1.1 and 1.3 g cm -5 respec­tively.

A third batch of 30 pots was filled with 2.5litres of nitrogen-free nutrient solution (pH 5.5)of the following composition ( 11M): K 2743, Ca1360, C1478, S 266, Mg 197, P 114, Na 108, Si66, Fe (as sequestrene) 12.8, B 9.0, Mn 8.0, Zn2.2, Cu 1.3, Mo 0.15 and Co 0.02.

Details of the preparation of stock nutrient solu­tions have previously been described (WanMohamad W. Othman 1983). Accurate volumesof each stock nutrient solution were dispensedinto the pots, using Jobling dispensers.

Planting and Management

Five phasey bean (Macroptilium lathyroidescv. Murray) seeds, scarified and inoculated withBradyrhizobium strain CB 756 (obtained fromThe Australian Estates Ltd) were sown on 31July, 1978, in pots of soil and sand, to a depth of1 to 1.5 cm. Seeds intended for solution culturewere placed in a tray of moist sand on the samedate, and were transplanted into the previouslyprepared nutrient solution when tne radic1eswere 2 to 3 cm long. The seedlings were held in2.5 cm diameter X ~.O cm deep plant supportbaskets filled with black polyethylene beads. Oneseedling was planted in each basket. The basketsfitted snugly into holes in pot covers made ofhigh density black polyethylene. The commer­cial Bradyrhizob,:um peat culture was added tothe nutrient solution one day before transplant­ing (approximately 6.7 X 10 5 viable cellsml -1). Initially, five seedlings were planted perpot, these being thinned to one plant per pot twoweeks later. Seedlings in the soil and sandcultures were thinned likewise.

136 PERTANIKA VOL. 9 NO.2, 1986

NODULATION AND GROWTH OF PHASEY BEAN

All pots were placed on two benches in anaturally lit glasshouse at University of Queens­land, Brisbane (Latitude 27° 28' S). Night tem­peratures in the glasshouse were maintainedbetween 18 and 22°C by placing four thermo­statically controlled electric heaters (each of 2.8kwatt) close to the benches. The meanmaximum temperature throughout ·the growthperiod was 27.2°C. The nutrient solution--wasaerated continuously with compressed air at therate of about 6 to 7 ml s - 1. Deionized water wasadded to the nutrient solution whenever neces­sary to ensure that the Toot systems were com­pletely submerged at all times. The nutrientsolution was maintained between pH 5.5 and 5.8by adding dilute (0.1 N) HCI or 0.1 M NaOHonce every six or seven days. Stock nutrient solu­tions were added to the pots of solution culture 6and 10 weeks after sowing without discarding theoriginal solution. The volumes of stock nutrientsolutions added on these two occasions were thesame as the initial volumes. The soil and sandcultures were supplied every second day with 150ml pot -1 of identical nitrogen-free nutrientsolution. The nutrient solution intended for soiland sand cultures was previously prepared in two25-litre polyethylene containers, using the

appropriate volumes of the stock solutionsdescribed earlier. The solid and sand cultureswere irrigated to field capacity with deionizedwater every other day. The diurnal variations intemperatureS of soil, sand and solution cultureswere recorded once in 8 to 10 days. The meandiurnal temperatures are presented in Table 1.

Plant Height and Non-destructive Nodule andLeaf Counts

Non-destructive nodule counts and otherobservations on plant growth and developmentwere made every two days on all plants grown insolution culture. Leaf number per plant was alsorecorded non-destructively every six or sevendays. Plant height was measured every two weekscommencing at Week 4. Since the number ofpots remaining decreased with time, the abovemeasurements and counts were made on pro­gressively decreasing plant numbers.

Harvesting

Four plants from each culture medium wereharvested every two weeks commencing four

TABLE 1Diurnal variations in temperature (OC) of air (in glasshouse), solution, sand and soil

Culture media

Time of day Air Solution Sand Soil

0800 25.0 18.5 19.0 18.0

1000 27.0 23.0 27.0 26.5

1200 28.0 26.0 30.5 28.5

1400 29.0 29.5 30.0 29.5

1600 26.5 31.0 29.0 28.5

1800 19.0 28.0 24.0 25.0+---

2000 21.0 26.0 21.0 22.0

2200 20.0 24.0 20.0 20.0

Arrow indicates the start of thermostatically controlled heaters.

The evening and night temperatures were 2 to 4 degrees higher in solution than the soil or sand culture. Ther~verse occurred at or before noon.

PERTANIKA VOL. 9 NO.2, 1986 137

WAN MOHAMAD WAN OTHMAN, C.J. ASHER AND L.R. HUMPHREYS

weeks after sowing (Week 4) and ending at Week14. Ripe pods, if any, were harvested before theyshattered. At harvest, the soil and sand werecarefully washed off the roots under runningwater, a sieve being placed underneath to collectany detached nodules or roots. All root systemswere thoroughly rinsed in deionized water andblotted dry. The plants were separated intoroots, nodules, stems (+ peduncles), -leaves,flowers and young pods, and, when present, ripepods. Ripe pods were further partitioned intohulls and seeds. Tap root length was measured,and the number of nodules, ripe pods, seeds,and leaves per plant were counted. The leaf areawas measured using an electronic leaf area meteror Paton Electronic Planimeter (Paton Indus­tries Pty. Ltd., Australia). All plant parts weredried in an oven for 48 hours at 80°C, and laterground in a hammer mill fitted with a 0.25 mmmesh screen.

Plant Analysis (total nitrogen)

Approximately 100 tq 200 mg of groundplant material was subjected to Kjeldahl diges­tion in a 75 ml digestion flask containing 5 ml ofconcentrated H ~O 4' <:me tablet (1 g) of seleniumcatalyst and 0.5 g sodium thiosulphate. Detailsof Kjeldahl digestion and the use of an auto­analyzer were described by A.O.A.C. (1965)and Gehrke et at. (1973) respectively. Exceptwhere otherwise stated, ground plant materialfrom each replication was bulked prior toanalysis to reduce the total number of samplesrequired for the assay.

RESULTS

Plant Growth

The length of tap root, plant height, leafarea and number increased with increasing plantage. In all culture media, rapid tap root elonga­tion occurred during early vegetative growth(Week 4 to 8) whereas leaf development andplant height increased rapidly between Week 8and 12 (Fig. 1), which coincided with the periodof active nodulation, except in sand culture inwhich active nodulation began from Weeks 10 to14 (Fig. 3a).

In solution and sand cultures, the primaryleaves showed mild nitrogen-deficiencysymptoms 1 to 2 weeks after planting, but thesymptoms disappeared approximately two weekslater. On the majority of plants, the first tri­foliate leaf emerged approximately four weeksafter planting. Leaf growth (leaf area andnumber) and plant height were initially better insoil than in sand or solution culture, but withtime (beyond Week 10) plants growing in solu­tion culture surpassed those in soil culture interms of leaf area, leaf number, and plant height(beyond Week 12). However, tap root develop­ment was always better in solution than soil orsand culture. During pod-filling and ripeningstage (Week 12 to 14) leaf area and number weresignificantly greater (P < 0.05) in solution thansoil culture; those in sand culture still laggedbehind these two treatments (Fig. 1).

Dry Matter A ccumulatz'on

In all culture media, dry matter accumula­tion in leaves, stems, roots + nodules, and in thewhole plant was gradual during the first eightweeks, but increased rapidly between Week 10and Week 12 for most plant parts with theexception of leaves in sand and soil cultures (Fig.2). In all treatments, flowering commencedbetween 9 and 10 weeks after planting. Themain feature of the flowering and early fruitingstage (Week 9 to 12) was the sudden increase inthe dry matter accumulation in stems anddeveloping fruits. At the end of this stage, theleaves and roots had attained their maximumgrowth. Although podding began soon afterflowering, active vegetative growth continueduntil Week 12 (with the exception of leaves insand and soil media), after which it remainedconstant. Abscission of some lower leaves occurr­ed during the pod-filling and ripening stages.Pods on the basal peduncles ripened rapidly. Insolution and soil cultures, ripe pods were themajOI: component of plant dry weight at finalharvest: However, in sand culture dry matteraccumulated largely in stems and to a smallerextent, in ripe pods. In all culture media, thenodulated roots and stems ceased growth duringthe pod-filling and ripening stage (Fig. 2).

138 PERTANIKA VOL. 9 NO.2, 1986

NODULATION AND GROWTH OF PHASEY BEAN

40 (a) Plant height 48 (b) Leaf numberA__ A

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Plant age (weeks after planting)Fig. 1: Effects of solution (l...), soil (.) and sand (.) cultures and plant age on plant height, leaf number,

leaf area, and tap root length (Vertical bars denote LSD's at P = 0.05)

PERTANIKA VOL. 9 NO.2, 1986 139

WAN MOHAMAD WAN OTHMAN, C.J. ASHER AND L.R. HUMPHREYS

r tEF l PR 1

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Fig. 2: Dry matter accumulation in vegetative and reproductive parts of phasey bean plants grown insolution (£.), soil (e) or sand (.) cultures (Stages of growth are denoted by: V = vegetative,EF = Flowering and early fruiting, PR = Pod-filling and rzpening. Vertical bars denote LSD's at

P = 0.01. Arrows indicate commencement of]lowering.)

140 PERTANIKA VOL. 9 NO.2, 1986

NODULATION AND GROWTH OF PHASEY BEAN

Over the first six weeks there were no signi­ficant differences in total dry matter productionamong the three culture systems. Between Weeks10 and 14, the total plant dry matter was signi­ficantly (P < 0.05) higher in soil than in sandculture. Differences between culture media werenot significant during vegetative growth. How­ever, beyond the tenth week, the plants in solu­tion culture grew much more rapidly than thosein soil culture. Plants in sand culture still laggedbehind those in soil culture. At week 14, theroots + nodules, stems, leaves and total drymatter production of solution-grown plants weresignificantly higher (P < 0.01) than those in soilculture (Fig. 2). At this plant age, the dry matteraccumulation in the plant parts and in the wholeplants was the lowest in sand culture. At finalharvest the pods (green and ripe) were the largestsingle component (46 to 52%) of plant dryweight in all treatments. Of the dry mattergained by the reproductive parts between Week10 to 14, 80 to 95% accumulated in ripe pods.The seed was the major component (appro­ximately 60%) of ripe pods.

Seed Yields

The mean weight of 1000 seeds (seed size)and total seed yield were significantly higher(P < 0.05) in solution than in soil culture (Table2). However, the other components of seed yieldwere not affected by either culture. The numberof ripe pods and seeds per plant, and seed yieldwere lower (P < 0.01) in sand than in soilculture, but the mean weight of 1000 seeds wassignificantly greater (P < 0.05) in sand culture.

Seed number per pod was not significantlyaffected by the culture medium.

Nodulatz"on

In solution culture, the first nodule wasvisible within 16 days of sowing. The first noduleoften formed on the tap root 5 to 6 cm below theplant support baskets. Newly formed noduleswere initially small and white in colour andturned pink within six to seven days. As the plantdeveloped, the pink nodules increased innumber and size. In all treatments there was asudden increase in nodule number betweenWeek 8 and 10 (prior to and during flowering),but nodule development was apparently retard­ed during the pod-filling stage as in the solutionand soil culture media (Fig. 3). Although nodulenumber was generally similar in all culturemedia, the mean weight per nodule (nodule size)and total nodule weight were significantly higherin solution culture than sand or soil culture.Total nodule dry weight and nodule size weresignificantly suppressed by sand culture,especially between Week 12 and 14.

In solution culture the roots were observedto be suberized, and the surface of nodulesdeveloped extensive lenticels (spongy tissues withlarge intercellular spaces). Nodules in soil andsand cultures developed relatively few lenticels.

Nitrogen Fixation and A ccumulation in PlantParts

In all culture media, nitrogen accumulationin vegetative parts increased in a sigmoid

TABLE 2Effects of solution, soil and sand cultures on components of seed yield (14 weeks after planting)

No. of ripe No. of No. of Mean wt. Total seed

Culture pods seeds seeds of 1000 yield

media plant -I pod -I plant -I seeds (g) (g plant -I)

Solution 31.8 19.8 630 8.71 5.49

Soil 32.0 18.6 595 7.66 4.56

Sand 7.7 19.4 150 9.00 1.35

LSD (P 0.05) 7.3 0.81 129 0.65 0.92

PERTANIKA VOL. 9 NO.2, 1986 141

WAN MOHAMAD WAN OTHMAN, C.J. ASHER AND L.R. HUMPHREYS

Plant ale (week~ after plantina)

Fig. 3: Effects oj solution (£,), soil (.) and sand

(.) cultures and plant age on nodule

number, nodule size and total nodule

dry weight (Vertical bars denote LSD sat P = 0.05.)

manner, but total plant nitrogen increasedexponentially from Week 4 to 14 (Fig. 4). Veryrapid increases in total plant nitrogen occurredduring the flowering and early fruiting stages.Prior to or during these stages the leaves andstems were the major sites of nitrogen accumu­lation. However, the pods gained substantialamounts of nitrogen during the reproductivestage. At Week 14 the pods (green + ripe) ofplants growing in solution, soil, and sand culturecontained 52%, 55 % and 51 % of the total plant

Effects of Nodulation and Nitrogen Fz'xationon Dry Matter Productz'on and Seed Yields

Plant growth was initially slow in solutionand sand cultures probably due to nitrogen star­vation before the establishment of effectivesymbiosis. The growth pattern of componentparts of phasey bean plants and the strong sinkeffect of pods for nitrogen and carbon (drymatter) were similar to those reported for cow­pea and soybean (Herridge 1977; Warembourget al., 1982). However, in phasey bean thegrowth of leaves, stems and roots declined verylittle during the pod-filling and ripening stage.This iack of decline in vegetative growth wasassociated with the continual increases in noduledry weight and in the rates of nitrogen fixationduring the period of rapid seed development(Figs. 2, 3c, 4f). Improved plant growth in solu­tion culture, relative to' the growth in othermedia, was apparently associated with increasednodulation and higher rates of nitrogen fixation(Figs. 2, 3, 4f). Results of the present experiment

DISCUSSION

nitrogen, respectively (equivalent to 372, 276and 106 mg N plant - 1). Of the nitrogen gainedby the reproductive parts at final harvest, 63 to69% ended up in seeds (Fz'g. 4d). During thereproductive growth stage, nodulated roots(roots + nodules) gained approximately 10 to20 % of total plant nitrogen.

Since nitrogen from seed source was verysmall (approximately 0.5 mg N seed -I), andthere was no deliberate application of nitrogenfertilizer (other than N naturally occurring insoil), total nitrogen in the whole plant (Fig. 4f)can be regarded as a close approximation tosymbiotic nitrogen fixation. Nitrogen fixationwas apparently unaffected by the various culturemedia during the vegetative growth. However,the rate of nitrogen fixation was significantlyhigher in solution than in sand culture, especial­ly during the reproductive stage (Fig. 4f).Although the rate of nitrogen fixation was con­sistently higher in solution than in soil cultureduring the reproductive growth stage, dif­ferences were significant only at Week 14.

14

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142 PERTANIKA VOL. 9 NO.2, 1986

NODULATION AND GROWTH OF PHASEY BEAN

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Fig. 4: Nitrogen accumulation in leaves, stems, pods, seeds, root and nodules, and whole plants in phaseybean plants grown in solution ( ~), soil (e) or sand (_) cultures. (Vertical bars denote LSD's atP = 0.05.)

PERTANIKA VOL. 9 NO.2, 1986 143

WAN MOHAMAD WAN OTHMAN, C.J. ASHER AND L.R. HUMPHREYS

support those of Franco and Munns (1982) whoshowed that nitrogen fixation (acetylene reduc­tion assay) and growth of French bean werebetter in solution culture than in gravel culture.

In the present study, the mean day/nighttemperatures (20/27 .2°C) in the glasshouse were3 to 5 degrees below the optimum requirementfor vegetative growth of phasey bean (Whiteman1968). Differences in air temperature have beenknown to cause marked changes in noduleweight and nodule size of cowpea (Dart andMercer 1965). It is highly unlikely that airtemperature was responsible for the differencesin nodulation and growth of phasey beanreported in the present study, since all plantswere subjected to the same ambient tempera­ture. The reasons for increased root growth andnodulation in solution culture are. not wellunderstood.

The slow nodule development during theinitial stages of plant growth (Fig. 3) is consistentwith the findings of other studies on otherlegumes, both in the field' and under controlledenvironmental conditions (Diatloff and Ferguson1970; Nicholas and Haydock 1971; Wilson1972). Nodule devel~pmentof Glycine was alsofound to be retarded in siliceous sand, and inyellow podzolic soil, taken from a similar site,compared to that in black earth (Thomas andWhiteman 1971).

The depressed nodulation in soil cultureduring the first few weeks of planting (Fig. 3) wasprobably due to the inhibitory effects of soilmineral nitrogen. Recent investigations indicat­ed that high levels of soil mineral nitrogensuppressed nitrogen fixation of other legumes(Alston and Graham 1982). There is evidence tosuggest that mineralization of soil nitrogendepresses nodulation of some legumes (Diatloff1967; Quilt and Dalal 1979) or replacessymbiotic nitrogen fixation of other legumes(AlIos and Bartholomew 1959).

The distinct development of lenticels on thesurface of nodules of solution-grown plants inthe present experiment, and the growth. ofmassive lateral roots of phasey bean under flood-

ed conditions in other studies (Siregar 1977;Chudasama 1981) are features associated withthe physiological adaptation of this plant to thewet culture system. Lenticels are often observedon nodules of other legumes growing in waterlogged conditions, and they are reportedlyassociated with the gaseous exchange in and outof nodules in a wet environment (Pankhurst andSprent 1975; Minchin and Summerfield 1976).

The data indicate that differences in rootenvironment (e.g. temperature, aeration, andthe physical nature of the rooting media) con­tribute to marked variations in nitrogen fixationwhich in turn affect vegetative growth and seedyields (Figs. 21, 41, Table 2). The 1000 seedweight, and seed number per pod obtained inthe present study are comparable with thosereported by Humphreys (1979). The markeddecline in total nitrogen in stems of solution­grown plants between Week 12 and 14, despitelarge increases in nitrogen fixation (Fig. 4b, d,j), was probably associated with the remoblliza­tion of nitrogen to the developing seeds whichwere produced in relatively large quantities insolution culture. With phasey bean there seemedto be little loss (remobilization) of nitrogen fromthe leaves during pod-filling stage (Fig. 4a). Itappears that high rates of nitf(?g~n fixation andthe partitioning of nitrogen into various plantparts are majpr factors influencing vegetativegrowth and reproductive development.

Benefits ofSolution Culture Techniques

Results of the present study indicate thatnitrogen-free solution culture enhances potentialnodule development, particularly nodule size(Fig. 3b) which makes nodule counting mucheasier. Harvesting and root-washing of solution­grown plants were completed within 1 to 2minutes per sample compared with 25 to 30minutes with the other media. One person wasable to wash 35 to 40 excised root systems withinone hour of harvest. With reference to acetylene

redUCtion assays, root incubation with acetylenecould be done quickly without having to staggerthe harvesting or incubation, thereby eliminat­ingdifferences due to diurnal fluctuations innitrogen fixation or nitrogenase activity.

144 PE:R.TANIKA VOL. 9 NO.2, 1986

NODULATION AND GROWTH OF PHASEY BEAN

Another major advantage of solution culture isthat nodulation and nodule development can beobserved and recorded non-destructively. Also,at harvest, all roots and nodules of solution­grown plants can be recovered easily. Thesefindings illustrate the suitability and importanceof solution culture as one of the experimentaltechniques in future experimental work, con­cemed with the accurate study of nodulationand nitrogen fixation of legume plants duringontogeny or following defoliation.

CONCLUSION

Nitrogen-free solution culture enhancednodulation largely by increasing nodule size.The rate of nitrogen accumulation in solution­grown plants was greater than that in plantsgrown in soil or sand culture. Sand culture sup­pressed nodulation, plant growth and seed yield.

During early plant growth, nitrogen accu­mulated largely in the leaves and stems, but podswere major sites of nitrogen accumulationduring pod-filling stage. Roots and nodules ofsolution-grown plants could be harvested easilyand rapidly without loss.

ACKNOWLEDGEMENTS

The authors would like to thank the Austra­lian Development Assistance Bureau for provid­ing the-necessary funds for this study. The finan­cial support was arranged through the technicalco-operation of the Australian Universities Inter­national Development Programme in collabora­tion with Universiti Pertanian Malaysia, thehome university of the senior author.

REFERENCES

ALLos. H.F. and BARTHOLOMEW, W.V. (1959):Replacem.ent of symbiotic fixation by availablenitrogen. Soil Sci. 87: 61- 6.

ALSTON, A.M. and GRAHAM, R.D. (1982): The in­fluence of soil nitrogen status and previous cropon nitrogen fixation (acetylene reduction) inBarrel Medics, Medicago transqtlata gaertn.Aust.J. Soil Res. 20: 51- 9.

ANDREW. C.S. (1976): Effects of calcium, pH andnitrogen on the growth and chemical compositionof some tropical and temperate pasture legumes.I. Nodulation and growth. Aust. J. Agric. Res.27: 611 - 23.

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(Received 13.fanuary, 1986)

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