Effect of cd and ni on soybean seed development

Effect of cd and ni on soybean seed development

Effects of the metal pollutants cadmium and nickel on

soybean seed development

H L Malan and J M Farrant*

Dept Botany, University of Cape Town, Private Bag, Rondebosch, 7701, South Africa

Abstract

The chloride salts of Cd or Ni were added to the nutrient

solution in which soybean (Glycine max) plants were

grown and the response of the plants to these pollutants

examined Both metals markedly reduced plant biomass

and seed production Accumulation was mostly in the

roots Nickel was more mobile than Cd, reaching higher

levels in all plant parts, especially seeds Within the

tissues of mature seeds, the highest concentrations of Ni

were found in the axis and testa The highest

concentrations of Cd were in the testa and cotyledon,

and the lowest in the axis When expressed on a per

seed basis, metal contents of these organs increased

with developmental age Nickel amounts were lower in

the pods than the seeds for all growth stages, however

there was no significant difference for Cd Cadmium

reduced mature seed mass This effect was mostly due

to decreased yields of lipids, protein and carbohydrates

Although the number of seeds per pod declined as a

response to Ni, seed mass was unaffected and there

was no apparent effect on storage reserves

Keywords: metal pollutants, cadmium, nickel, heavy

metal, Glycine max, soybean, seed

Introduction

There are several metal pollutants that are considered

to be of potential threat to environmental systems

These include Cd, Cr, Cu, Hg, Ni, Zn and Pb

(Marschner, 1982; Friedland, 1990) Due to their

distinct chemistry and characteristics, each represents

a rather different hazard to the environment In this

study, the effect of Cd and Ni on the development of

soybean seeds was examined

Cd is a non-essential element in plants (Verkleij

and Schat, 1990) It is recognized as one of the most

potentially hazardous of all metal pollutants since it is

extremely toxic to humans and other animals (Rascio

et al., 1993; Cieslinski et al., 1996) and is known to

accumulate in mammalian kidneys (Quaife, 1981) Exposure is due mainly to high amounts in the diet, although tobacco smoking and occupational exposure

to CdO fumes are also important sources (Alloway, 1990) The fact that this metal is fairly readily taken up

by plants and translocated to aerial organs facilitates its entry into the food chain (Rauser and Meuwly 1995;

Salim et al., 1995).

Nickel, on the other hand, although a serious

environmental pollutant (Sajwan et al., 1996) and phytotoxic at high concentrations (L’Huillier et al.,

1996) is considerably less toxic to living organisms than Cd It has been found by some researchers to be

an essential micronutrient in certain plant species, especially when grown on urea-based media (Breckle, 1991; Gerendas and Sattelmacher, 1997) In comparison to Cd, Ni is even more mobile within plants (Marschner, 1982)

Natural amounts of Cd in the environment are generally low, however anthropogenic activities can drastically increase these levels (Woolhouse, 1982) Such activities include: zinc mining and smelting, use

of sewage sludge for agricultural fertilization, motoring (car exhaust fumes), combustion of fossil fuels, application of phosphate fertilizers, industrial and manufacturing processes (Lund, 1981; Xian, 1989;

Rascio et al., 1993; Marchiol et al., 1996).

Nickel is generally more naturally abundant than

Cd Some native soils, specifically mafic and ultramafic (serpentine) soils, have high indigenous amounts of this element (Mishra and Kar, 1974; Steyn

et al., 1996) Specially adapted species and populations

of plants have evolved to survive these conditions (Peterson, 1983) Localized high contents do occur as a result of mining, burning of fossil fuels, fertilizer application, automobiles (McIlveen and Negusanti, 1994) and industrial activities such as the manufacture

of Ni-steel alloys (stainless steel), electronic components and batteries (McGrath and Smith, 1990) Since the late 1960s extensive research has been carried out on the threat posed by metal pollutants to the environment (Marschner, 1982; Tjell and Christensen, 1992) However, very little research has

*Correspondence

E-mail: hmalan@botzoo.uct.ac.za; farrant@botzoo.uct.ac.za

been carried out on the effect of metal pollutants on

seed development (Siegel and Siegel, 1985) A database

survey on papers concerned with uptake, accumulation

and translocation of heavy metals by vascular plants,

revealed that fewer than 11% of the almost 25 000 listed

papers studied the effect of metals on reproductive

parts (Nellessen and Fletcher, 1993) Research that has

been carried out has been limited almost exclusively to

analyses of total metal content within seeds and

whether this poses a potential risk to consumers There

are few reports concerning the effect metal pollutants

may exert on metabolic and developmental processes

occurring within the seed Soybeans, because of their

high nutritive value, are an important agricultural crop,

grown increasingly in developing countries as a food

source (Gupta, 1983; Odendaal et al., 1984) Thus it is

important to assess the impact of metal pollution on

seed production, and in this study we examined the

effect of Ni and Cd on soybean seed development

Materials and methods

Seeds were harvested from soybean plants (Glycine

max (L.) Merr cv Crawford), grown in a modified

Hoagland’s nutrient solution amended with either Ni

or Cd Details for the production of these seeds are

given below

Plant cultivation and seed production

Plants were grown in a controlled environment

chamber at a 258C day and 208C night temperature,

12-h photoperiod and PAR of 800 µmol m22s21 Seven

days after germination, seedlings were transferred to

one litre plastic jars filled with nutrient solution The

concentration of macronutrients was as follows: 1 mM

KH2PO4, 2 mM MgSO4.7H2O, 4 mM CaNO3, 4 mM

KNO3and micronutrients: 89.9 µMFeNaEDTA, 46 µM

H3BO3, 9.1 µM MnCl2.4H2O, 0.8 µM ZnSO4.7H2O,

0.3 µM CuSO4.5H2O, 0.1 µM H2MoO4.H2O After two

weeks in the starting jars, seedlings were transferred

to a circulating nutrient system, which consisted of a

25-litre growth tank, in which the roots were

immersed This was attached by tubing to a reserve

tank Four plants were allocated to each growth tank

and the total volume of nutrient solution was 40 litres

The pH of each circulating system was adjusted to 6.0

every 1–2 days and deionized water added to bring

the total volume back to 40 litres Fresh nutrient

solution was made up every 10 days and the growth

tanks were constantly aerated The same composition

of nutrient solution was used as in the starting

containers except that the chloride salts of either Cd or

Ni were added to give resulting metal pollutant

concentrations of 0 mg/litre (control) 0.05 mg/litre Cd

or 1 mg/litre Ni The Cd-stress experiments were first

carried out utilizing one growth tank as the control treatment and another three tanks for the metal treatment Subsequently the Ni-stress experiments were conducted using the same tanks as control and treatment tanks Care was taken to ensure that the environmental and growth parameters were constant

In addition, the growth tanks were washed after the Cd-stress experiments and the rinse water analysed for this metal using the standard procedure (see below) Cadmium contamination of the growth tanks was minimal and is therefore not discussed further Seeds were harvested at four distinct stages in development which were determined by the size and morphology of the pod and seed Pods were measured with regard to length, depth and thickness, as well as the extent to which the depth of the locule was filled

by the developing ovule This was based on the

method of Miles et al (1988) Approximate DAF (days

after flowering) for each stage are also given

Immature pods (IP) – pods dark green in colour, at least 50 mm in length and 10 mm in depth Ovules 4–6 mm in depth, i.e filling half the depth of the locule Seeds in rapid growth stage DAF approxi-mately 5 16–17

Expanded pods (EP) – pods light green in colour, fully expanded (>7 mm in thickness) and turgid Ovules filling the entire locule depth, green and showing no yellowing Seeds at mid-seed fill period DAF approximately 5 30

Yellow pods (YP) – pods light yellow in colour and pliable Seeds detached from the funiculus, soft and bean shaped Seeds physiologically mature Maturation drying in progress DAF approximately 5 51

Brown pods (BP) – pods light brown in colour and brittle Seeds mature, dry, hard and round in shape DAF approximately 5 54

Seeds were harvested at each growth stage, freeze-dried for 48 h and stored at 2808C until further processing

Uptake of metal pollutants

Plants were grown in nutrient solution amended with either 0.05 mg/litre Cd or 1 mg/litre Ni and the visual toxicity symptoms noted At senescence (when all remaining pods were at the BP stage), plants were separated into roots, leaves and pods These were washed under running deionized water for 20 s and oven-dried at 708C for 48 h Material was finely ground, and 0.5-g samples ashed in a muffle furnace for 5 h at 5008C Freeze-dried seed samples were processed in a similar manner, except that the oven drying step was omitted and 2-g samples were used All samples were then digested for 24 h in concentrated HNO3 at a temperature of 1508C and made up to a final volume of 25 ml in 0.1 M HNO

Samples were analysed for Cd or Ni using a Jobin

Yvon JY138 ultratrace ICP-AES (inductively coupled

plasma - atomic emission spectrophotometer)

The effect of metal pollutants on seed development

Seeds, harvested from metal-treated plants at various

stages of development, were compared with those

from control plants The following parameters were

examined: total seed yield, mass, moisture content,

germination and storage reserve accumulation Lipid

determination was carried out on freshly harvested

seeds according to a modified method of Christie

(1973) Total extractable carbohydrates were

determined on freeze-dried tissue according to the

method of Adams et al (1980) Total N (nitrogen) was

assayed using the standard micro-Kjeldahl method

(Stock and Lewis, 1986) and the crude protein content

estimated by multiplying the nitrogen content by a

factor of 5.49 which is appropriate for soybean seeds

(Mossé and Pernollet, 1983)

Statistical treatment of the data

Significant differences between means were examined

using Student’s t test at the 95% confidence limit In

cases where sample size was small, Wilcoxon’s rank

sum test was employed

Results

Visual toxicity symptoms

Visual toxicity symptoms exhibited by the leaves on

exposure to the two metals were similar to those

previously described for white beans by Rauser (1978)

The pods and seeds produced by metal-treated plants

were for the most part indistinguishable in appearance

from those of the controls However, plants treated

with Ni occasionally produced deformed terminal

racemes, composed of pods greatly reduced in size

(approximately 10 mm compared to 50 mm) These

abnormal pods remained green and contained either

no seeds or those that were rudimentary and

non-viable

Effect on plant biomass

Table 1 shows the effects of Cd and Ni on plant growth

parameters as represented by pod yields as well as by

the root dry mass per plant Both metals markedly

decreased pod production and root biomass relative to

the controls Cadmium appears to be more toxic than

Ni since a lower concentration of the former was

required to elicit the same degree of pod and root

biomass depression

Distribution of metal within the plant

Table 2 shows the Cd and Ni content of roots, leaves, pods and mature (BP) seeds taken from metal-treated and control plants Metal content in all parts was higher than in the equivalent organ of controls Considerable Cd enrichment occurred in the roots, accumulating to a concentration of 130 µg/g dm from the 0.05 mg/litre present in the nutrient solution Cadmium values for the aerial portions of the plant were low in comparison to the roots, the concentration

in the leaves being 30-fold lower than the roots Cadmium contents were lowest in the reproductive tissues Nickel was also concentrated in the roots with lower levels in the shoots and seeds Nickel values for the leaves were 20-fold less than for the roots In general the amounts in all parts were much higher than for Cd Leaves and seeds accumulated similar Ni concentrations but pod contents were considerably lower

The distribution of Cd and Ni within mature soybean seeds is shown in Table 3 Cadmium enrichment occurred in the testa and cotyledons with very little accumulating in the axis Nickel concen-trations on the other hand were highest in the axis, intermediate in the testa and lowest in the cotyledons

The effect of seed growth stage on metal accumulation

The concentration of the two metals in seeds and pods

at each developmental growth stage was examined (Table 4) Concentrations were always significantly higher in treated, relative to control, seeds and pods For all developmental stages Ni contents were higher

in the seed than in the pod However, there appeared

to be little difference between pods and seeds of Cd-treated plants whatever the stage of development Metal concentrations (calculated per gram dry mass) were higher in young (IP) seeds but declined significantly by the EP stage However if the results are expressed on a per seed basis, metal content of treated seeds generally positively correlated with seed age

The effect of metal pollutants on seed growth parameters

Table 5 summarizes the effect of Cd and Ni on seed mass and the average number of seeds per pod for mature seeds The seed number did not change during development and so results for earlier developmental stages are not given Cadmium had a significant effect

on the average size of BP seeds, resulting in decreased

seed mass relative to the controls (P≥0.001) However this metal did not affect the average number of seeds per pod On the other hand, although Ni did not exert

mg/litre Cd or 1 mg/litre Ni SD given in parenthesis n 5 3 for metal treatments, n 5 4 for control

Growth parameter Cd-treated plants Ni-treated plants Control

No pods/plant 67.5 (6 13.3) 68.7 (6 9.1) 93.5 (6 7.20) Root dry mass (g) 6.8 (6 3.3) 6.9 (6 0.12) 10.6 (6 2.1)

Table 2.Distribution of Cd and Ni in various parts of plants grown in 0.05 mg/litre Cd or 1 mg/litre Ni SD given in parenthesis Minimum sample size 5 3

Cadmium (mg/g dm) Nickel (mg/g dm) Plant part

Roots 130.09 (6 34.2) 1.31 (6 0.19) 1100 (6 40.0) 3.1 (6 0.1) Leaves 3.80 (6 0.08) 0.43 (6 0.17) 48.1 (6 1.5) ND Pods 0.78 (6 0.24) 0.48 (6 0.01) 12.5 (6 0.69) ND Mature seeds 0.96 (6 0.15) 0.12 (6 0.04) 49.1 (6 5.75) 0.2 (6 0.01)

Table 3. Distribution of Cd and Ni within the tissues of mature (BP) soybean seeds harvested from plants grown in 0.05 mg/litre Cd or 1 mg/litre Ni Because of the low amounts of metal present in the seed, the dry mass of tissue required per sample was high and thus sample size was small (n 5 2 for axes, n 5 3 for other tissues) The approximate number of seeds required per sample is given for each treatment The same mass for treatment and the equivalent control was used SD given in parenthesis

Cadmium ( mg/g dm) Nickel ( mg/g dm) Seed tissue

Treatment Control seeds/ Treatment Control seeds/

Testa 1.52 ( 6 0.51) 0.04 ( 6 0.01) 40 77 ( 6 3.0) ND 20 Cotyledon 1.53 ( 6 0.19) 0.05 ( 6 0.01) 15 55.7 ( 6 1.9) ND 15 Axis 0.04 ( 6 0.06) 0.01 ( 6 0.00) 80 99.2 ( 6 3.4) 0.98 ( 6 1.2) 50

Table 4. Effect of seed development stage on metal concentration in seeds and pods harvested from plants grown in 0.05 mg/litre Cd or 1 mg/litre Ni Seed concentrations given both on a mg/g dm and per seed basis IP 5 immature pod, EP 5 expanded pod,

YP 5 yellow pod, BP 5 brown pod Complete descriptions of developmental stages given under Materials and methods Ni control values omitted for clarity as all were below detection limit SD given in parenthesis Minimum sample size = 3

(6 0.01) (6 0.03) (6 0.02) (6 0.12) (6 0.00) (6 5.8) (6 0.5) (6 0.01)

( 6 0.01) (6 0.06) (6 0.29) (6 0.09) (6 0.01) (6 1.2) ( 6 5.2) (6 0.74)

(6 0.02) (6 0.04) (6 0.15) (6 0.07) (6 0.02) (6 1.8) (6 2.3) (6 0.44)

( 6 0.01) (6 0.01) (6 0.24) (6 0.15) (6 0.03) (6 0.69) (6 5.8) (6 1.14)

a significant effect on seed mass, the presence of this

element in the growth medium did decrease the mean

number of seeds per pod (p≥0.001)

The effect of metal pollutants on storage reserves

Figures 1, 2 and 3 show the effect of Cd and Ni on

storage reserves of treated seeds Protein contents, as

determined from total nitrogen, increased with seed

development in all treatments (Fig 1) Cadmium

significantly reduced the total protein content of

mature seeds, a reflection of reduced seed mass

Nickel on the other hand did not result in any

significant changes in protein content

Lipid content increased with seed age until the YP

stage, thereafter it levelled off (Fig 2) In the case of

the Cd treated seeds and control seeds, lipid levels

were slightly lower than at the YP growth stage due to

the fact that the BP seeds were slightly smaller in size

(data not shown) Cadmium significantly reduced the

lipid content of the mature seeds relative to the

controls, again a reflection of reduced seed size A

similar effect was evident in Ni-treated mature seeds

However the effect of this metal was not statistically

significant

Soluble carbohydrate (sugars) and insoluble

carbohydrate (starch) levels are given in Figure 3

Soluble carbohydrates increased with seed

development, reaching a peak at maturity Starch, on

the other hand, increased during the early growth

stages (IP and EP) and then declined in mature seeds

Cadmium decreased carbohydrate levels in treated

compared to control seeds, although only the effect on

starch was significant Nickel decreased the levels of

soluble sugars compared to the controls

Discussion

The major effect exerted by Cd and Ni in this study

was a general reduction in plant biomass This was

observed in the form of decreased root mass and

decline in pod yield and was the response to both

Table 5.Effect of Cd and Ni on dry mass and seeds per pod for mature (BP) seeds harvested from plants grown in 0.05 mg/litre Cd or 1 mg/litre Ni SD and sample size given in parenthesis

parameter Treatment Control Treatment Control

(6 0.4, n 5 14) (6 0.19, n 5 11) (6 0.4, n 5 24) (6 0.2, n 5 20) Seed mass (g/seed) 0.192* 0.229 0.198 0.193

(6 0.04, n 5 84) (6 0.02, n 5 53) (6 0.04, n 5 37) (6 0.05, n 5 31)

* Indicates significance at P> 0.001

Figure 1.Effect of Cd and Ni on protein content (mg/seed)

of seeds harvested from plants grown in 0.05 mg/litre Cd or

1 mg/litre Ni IP and YP values for Ni and Ni control treatments not determined (■ Cd treatment, ❒ Cd control, vertical stripes 5 Ni treatment, horizontal stripes 5 Ni control) n 5 3.

Figure 2.Effect of Cd and Ni on lipid content (mg/seed) of seeds harvested from plants grown in 0.05 mg/litre Cd or 1 mg/litre Ni IP and YP values for Ni and Ni control treatments not determined (■ Cd treatment, ❒ Cd control, vertical stripes 5 Ni treatment, horizontal stripes 5 Ni control) n 5 3.

metal pollutants Reduction in plant biomass as a

result of heavy metal stress appears to be an almost

universal finding (MacNicol and Beckett, 1985; Leita et

al., 1993; Dudka et al., 1996; Ouzounidou et al., 1997) A

few cases of yield enhancement due to metal

pollutants have been reported in the literature, but

these were from experiments utilizing extremely low

concentrations of metals (Mishra and Kar, 1974;

Breckle, 1991) Root growth appears to be especially

susceptible to metal toxins compared to the shoots and

has been used extensively as a convenient criterion of

metal tolerance (Ouzounidou et al., 1997) A reduction

in the yield of reproductive tissues has also been

reported for several species (Huang et al., 1974;

Cimino and Toscano, 1993; Singal et al., 1995).

Nickel concentrations in seeds were consistently

higher than those in pods for all growth stages, whilst

there was little difference in the Cd content of the two

This suggests that the pods pose only a minimal

barrier, and exert little screening effect on metal

pollutants Other reports in the literature however do

not support these findings Cimino and Toscano (1993) examined uptake of Cd, Pb and Cu from sludge- or metal-amended soils into pea and bean seeds Cadmium contents of the pods were significantly higher than of the seeds for both species Haghiri (1973), experimenting with radioactive Cd in soybean plants, also found that Cd was higher in pods than seeds It is possible that the pod to seed ratio may be dependent on the concentration of Cd supplied to the plant

The low Cd content of seeds found in this study is similar to other values found in the literature, and is consistent with the general view that plant reproductive organs tend to be protected from toxic metals (Marschner, 1982) On the other hand, high seed concentrations of Ni have also been reported by

other authors (Halstead et al., 1969; Cataldo et al., 1978)

and support the contention that Ni appears to be an exception to this rule of minimal seed accumulation

(Welch, 1995; Sajwan et al., 1996) Thus Ni appears to

be more mobile within plants than Cd, as shown by the elevated Ni concentrations of this element in all plant parts Whilst the concentration of Ni used in the nutrient solution was twenty times higher than that of

Cd, calculation of the concentration factor (i.e the ratio of the concentration of metal accumulated to that available for uptake) for seeds in this study, yields values of 20 for Cd and 50 for Ni Thus the magnitude

of accumulation in soybean seeds was greater for Ni than for Cd, and was not simply a result of a higher supplied concentration

Many variables such as soil composition, temperature, pH, chemical form and concentration have been shown to affect plant uptake of metal pollutants in the field (Ernst, 1996) In this study, plants were grown in nutrient solution and the root environment strictly controlled Extrapolation of results from plants grown in such an artificial system

to those in the field can be difficult In the soil, due to binding of metal cations by soil components not all the metal is available for plant uptake (Chaney, 1991) In nutrient solution systems, on the other hand, the proportion of bioavailable metal ions is often higher because of the absence of this binding and thus plant uptake is often greater from nutrient solution than from a soil containing the equivalent concentration of

a given metal ion Reports of Cd values slightly higher than 1 mg/g dm have been reported in the literature for seeds harvested from plants grown in polluted

areas (Yoshida, 1986; Stefanov et al., 1995) Therefore it

is felt that the levels of metal pollutants used in this study and the effects exerted by them are comparable

to those that may be found at contaminated sites in the field It is of interest that the limit for Cd in legume crops as recommended by the World Health Organization in 1992 (Petterson and Harris, 1995) is 0.1 mg/g dm

Figure 3.Effect of Cd and Ni on the sugar and starch content

(mg glucose/seed) of seeds harvested from plants grown in

0.05 mg/litre Cd or 1 mg/litre Ni IP and YP values for Ni

and Ni control treatments not determined (■ Cd treatment,

❒ Cd control, vertical stripes 5 Ni treatment, horizontal

stripes 5 Ni control) Minimum n 5 2.

Both metal treatments resulted in declined pod

numbers, which in turn affected total seed yield Thus

the primary effect of these metals was on early events

such as flower production or fruit set Once committed

to pod formation however, the pollutants had

differing effects on seed development Nickel

treatment resulted in reduced numbers of seeds per

pod, but seed mass was equivalent to control seeds

Cadmium treatment resulted in the same number of

seeds per pod as the control, but individual seeds

were smaller This could be explained in terms of

photosynthate available from the parent for reserve

accumulation Because Ni treatment reduced the

number of seeds during early development, there was

more photosynthate available per seed for reserve

accumulation With greater numbers of seeds reaching

the stage of nutrient deposition, Cd treatment resulted

in reduced storage reserve accumulation and this

affected seed mass Although the total concentration

of Cd in the seeds was low, 83% was located in the

cotyledon, the principal site of storage reserve

deposition On the other hand, only 43% of the Ni

taken up into the seeds was located in the cotyledons

This may be the reason that reserves are lowered in

seeds exposed to Cd, but reserve accumulation and

hence seed size were not affected by Ni Cieslinski et

al (1996) concluded that yield reduction in strawberry

fruit when grown in Cd-amended soil resulted mainly

from decreases in fruit number rather than average

weight per berry Moraghan (1993) when investigating

the effect of the same metal pollutant found similar

effects on yield parameters of flaxseed On the other

hand, Singal et al (1995), examined the effect of Cd on

seed mass of fenugreek, and found that at all

developmental stages there was a general decrease in

seed size with increasing Cd concentration

The differing metal distribution patterns found in

the seeds is interesting When expressed as percentage

of total metal uptake, 42% of the total Ni was localized

in the maternal tissue of the testa and is unlikely to

affect subsequent germination Fifteen percent was

found in the axis However, despite this relatively high

value, it was found (data not reported) that seed

germination was not profoundly affected, vigour was

slightly decreased relative to the controls but viability

was the same In the case of Cd, although the total

concentration of the metal in the seed was low, 83%

was located in the cotyledon and comparatively little

in the testa (17%) or axis (0.1%) Even though the

amount in the axis was extremely low, vigour was also

slightly decreased in these seeds, but there was no

effect on viability Thus, the results indicate that Cd is

more toxic than Ni and exerts a more pronounced

effect on seed development

It is possible that the quality of the storage reserves

within the seed are altered as a response to the

presence of either metal pollutant, since only the

quantities of storage reserves were investigated in the

present study Stefanov et al (1995) found that lead

altered the lipid content in seeds of green pepper, shifting the balance between saturated and unsaturated fatty acids in a complex manner Cadmium was found to generally increase lipid phosphorus (P) and decrease protein P in the seeds of

fenugreek (Singal et al., 1995) Further studies on the

effect of toxic metals on the chemical composition of soybean seeds may be rewarding, especially if the nutritional value is affected Although it is well documented that soybean seeds contain very little

starch at maturity (Adams et al., 1980) results from

these experiments consistently showed starch contents

up to 20 mg/seed in the oldest growth stage This may

be due to the cultivar, but is most likely due to inefficient separation of soluble sugars from starch during the extraction process

In conclusion, it can be seen that the presence of metal pollutants in the nutrient solution greatly affected the parent plant At levels of Cd or Ni where adult plants could survive, seed production was greatly diminished Significant amounts of metal did enter the seeds, especially in the case of Ni due to its enhanced mobility This did not markedly reduce the quality of the seed with respect to the quantity of nutrients in the case of Ni, however yields of storage reserves from Cd-treated plants were reduced Cadmium appears to have a more profound effect on seed development than Ni

Acknowledgements

This work was supported by the FRD (Foundation for Research Development), South Africa

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Received 22 January 1998, accepted after revision 10 July 1998

© CAB INTERNATIONAL, 1998