African Crop Science Journal African Crop Science Society ISSN: 1021-9730 EISSN: 2072-6589 Vol. 8, Num. 3, 2000, pp. 283-294

African Crop Science Journal, Vol. 8. No. 3, pp. 283-294

Cowpea Seed Coat Chemical Analysis in Relation to Storage Seed Quality

E. A. Asiedu, A. A. Powell¹ and T. Stuchbury¹
Crops Research Institute, P. O. Box 3785, Kumasi, Ghana
¹Department of Agriculture, McRobert Building, Aberdeen, AB24 5UA, Scotland

(Received 24 March, 1998; accepted 3 April, 2000)

Code Numberl CS00030

INTRODUCTION

Imbibition is an essential first step towards the seed hydration required for initiation of biochemical changes that lead to germination. However, uptake of water can cause imbibition damage to the embryos of germinating seeds, particularly those that imbibe water quickly, and consequently this may cause reduction in seedling emergence. Imbibition damage results from the rapid inrush of water into the embronic cell of fast imbibing legume seeds, leading to physical disruption of the cell membrane so that cellular structure is impaired. Imbibition damage was first recognised in a study when pea seeds were imbibed in free water with the testae intact or removed (Powell and Matthews, 1978). Subsequent vital staining revealed differences in damage to the abaxial surface of the cotyledons.

In some species of legumes, susceptibility to imbibition damage is associated with differences in seed coat colour. In dwarf French bean, cultivars with white seed coats imbibed water more rapidly than pigmented ones and showed higher levels of imbibition damage as evident by high leachate conductivity and lower percentages of complete vital staining of embryos (using 2, 3, 5 triphenyl tetrazolium chloride) as well as reduced germination when compared to brown and black cultivars (Powell et al., 1986a; 1986b). Similar observations have been made in pigmented chickpea and cowpea (Legesse, 1991; Legesse and Powell, 1992; Asiedu and Powell, 1998). The vigour differences between unpigmented and pigmented cultivars resulted in differences in field emergence and hence differences in grain yield of snap bean (Deakin, 1974) and chickpea (Knight and Mailer, 1989).

Differences in seed coat colour have been associated with differences in the composition of chemical compounds in the seed coats of leguminous species. Chemical compounds found in seed coats of legumes include tannins, lignin and non-tannin polyphenolic compounds (which comprises several groups of phenolic compounds). The concentrations of these compounds may differ depending on the level of pigmentation in the seed coat. For example, Carmona et al. (1991) found higher amounts of tannins in a black-seeded bean (2.48 tannic acid equivalent; TAE) than in a white cultivar (0.54 TAE). Also the tannin content of faba bean seed coats determined by the protein precipitation method showed high amounts of tannin in pigmented cultivars and low amounts in unpigmented cultivars (Bos and Jetten, 1989). Similarly, differences in tannin content have been found between pigmented and unpigmented seed coats of faba bean (Marquardt et al., 1978; Bond and Smith, 1989; Cabrela et al., 1989; Garrido et al., 1989, van der Poel et al., 1991 ) and Phaseolus vulgaris (Bressani and Elias, 1989).

In pigmented cultivars of legumes, other compounds such as flavonoids (including colour pigments, anthocyanins) and lignins that may be present in their seed coats contribute to colour formation. Kannenberg and Allard (1960) and Nozzolio et al. (1988) observed that the coloured seed coats also contained 15 times more lignin than the white seed coats. Recently, Morrison, et al. (1995) observed twice more lignin in pigmented seeds of cowpea when compared to unpigmented ones.

This study had two objectives. First, to compare changes in germination and the incidence of imbibition damage between pigmented and unpigmented cultivars of cowpea (Vigna unguiculata L. Walp) during storage. Second, to determine if differences in chemical composition exist between pigmented and unpigmented seed coats of cowpea and if so, determine if the differences can be related to the physiological deterioration of these seeds during storage.

MATERIALS AND METHODS

Seeds of five cowpea cultivars differing in seed coat colour originally developed at the International Institute of Tropical Agriculture (IITA) in Ibadan, Nigeria, were produced in Ghana between July and September, 1992 and used to conduct the experiment in Aberdeen, Scotland. These were IT83S-818 (white, black eye), IT81D-1137 (white, brown eye), TVX 3236 (cream & brown), TVX 2724-01F, (brown) and IT82D-32 (dark brown). The seeds were subjected to various forms of ageing, including controlled deterioration, accelerated ageing and simulated tropical storage, and parameters for seed quality were determined. The association of these parameters to the concentration of seed coat chemical extracted from each cultivar was analysed.

Controlled deterioration. Seeds of three cultivars of cowpea, {IT83S-818 (white, black eye), TVX 2724-01F (brown) and IT82D-32 (dark brown)} were subjected to controlled deterioration using the method described by Matthews (1980) and Powell and Matthews (1981b) before they were tested for germination. The seed moisture contents were adjusted to 20% by imibibing these seeds slowly in damp jay cloth to the desired weight equivalent to this moisture content. Four sets of each cultivar corresponding to four ageing periods, each containing 100 seeds, were counted into laminated aluminium foil packets. The packets were heat sealed, equilibrated at 10°C for 24 hours and three sets of packets incubated at 40°C for 1, 2 and 4 days. After each period of incubation, seeds were dried back to their original moisture contents before testing for germination (radicle emergence) and the results compared to the control treatment (unaged). The control treatment (the 4^th set) had their moisture contents raised to 20%, equilibrated at 10°C for 24 hours and then dried back and tested for germination without being held at 40°C.

Accelerated ageing and simulated tropical storage. The accelerated ageing test was conducted using a procedure similar to that described by Delouche and Baskin (1973) and TeKrony (1995). Seeds of five cowpea cultivars were aged in desiccators at a storage temperature of 40oC and relative humidity of 100%. Duplicates of six sets of 100 seeds of each cultivar corre-sponding to each ageing period, were counted into muslin bags (measuring 6 cm x 6 cm) and stored in two desiccators which were then covered tightly with their lids. Samples of seed were taken at 48 hour intervals for 6 days and tested for germination, electrical conductivity of seed leachate, vital staining of cotyledons and imbibition rate, and results compared to unaged seeds.

A more prolonged storage experiment was conducted under simulated tropical conditions at 30oC and 75.5% relative humidity for six months. The storage relative humidity was attained using saturated sodium chloride (NaCl) in tightly covered desiccators, a method similar to that described by Winston and Bates (1960). The desiccators were allowed to equilibrate for 7 days in an incubator set at 30oC before storing the seeds. Duplicates of six sets of 150 seeds for each of the five cultivars were stored in muslin bags (6 cm x 6 cm) and one bag of each cultivar removed at 30 day intervals to determine percentage germination, leachate conductivity, percentage vital staining of cotyledons and imbibition rate, and the results were compared to unstored seeds.

Germination test. Four replicates of 25 seeds were germinated between moist paper towels with two paper towels beneath and one above. Seeds were arrangeed linearly midway between the two long edges of the paper towels. The paper towels were then rolled and placed upright in small plastic containers, a third filled with water. These were packed in plastic trays and each tray covered with a polythene bag to reduce loss of moisture from the paper towels and trays placed in an incubator at 25-32°C. First and final germination counts were taken on the fourth and eighth day, respectively, in the cases of the accelerated ageing and the simulated tropical storage experiments. In both cases, seeds were considered germinated if the seedlings were normal as described by AOSA (1981). In the controlled deterioration experiment, however, daily counts of radicle emergence were taken up to eight days. Seeds with 5 mm radicle emergence were considered germinated.

Measurement of electrical conductivity of seed leachate. Electrical conductivity of the seed leachate was determined for twenty individual weighed seeds to which 4 ml deionised water had been added 24 hours earlier for imbibition to take place. The measurement was done under laboratory condition (20°C), using the automatic seed analyser (ASAC) model 1000. This instrument measures the electrical current passing through the water as a result of the leakage of electrolytes from weak or damaged embryonic cells into the surrounding water. The measurement was expressed in micro-amps per gram of seed (µamps g^-1).

Measurement of imbibition rates of seeds. Imbibition rates of ten seeds of each cultivar were measured in two replicates. Seeds were initially weighed individually before being placed in a cell of an automatic seed analyser (ASAC) to which 4 ml deionised water at 20oC was added. Each seed was removed, wiped with a tissue, weighed and returned to its cell at hourly intervals for six hours. The rate of imbibition was expressed as a percentage increase in weight at the end of each hour compared to the initial weight before imbibition. The measurement of the rate of imbibition was carried out at a room temperature of 20oC.

Vital staining of seed embryo. The living tissues of the seed’s embryo were stained using 2,3,5 triphenyl tetrazolium chloride (TTC) as described by Powell and Matthews (1979) after seeds had imbibed water for 24 hours. Four millilitres of 1% (w/v) TTC solution in distilled water was added to each seed in a cell after the testa had been removed. The seeds were then held in the dark for 3 hours at 30oC. In living tissues, the TTC reacts with the dehydrogenase enzymes in the cotyledons to produce a red stain called formazan (Cottrell, 1948; Roberts, 1951). Tissues of the cotyledons damaged during imbibition remain unstained. Cotyledons were then categorised as (1) x= 100, (2) 100>x>50, (3) 50>x>1 or (4) x= 0% stained, where x stands for the extent of staining of each embryo. The percentage of each category was then calculated for each period of storage.

Seed coat chemical analysis. Chemical pigments were extracted from the seed coats of the five cowpea samples using the modification of the procedure described by Carmona et al. (1991). This was done after the seed coats had been manually removed from the dry seeds and ground to less than 1 mm using an electric mill. Ground seed coat (2.5 g) of each cowpea sample was extracted in 25 ml of 1% HCl in methanol by shaking at 5 minute intervals for 30 minutes at room temperature (20-22oC). The supernatants of the extracts were filtered through Whatman No. 1 filter paper and rotary-evaporated to dryness at 35oC. The dried concentrate was redissolved in 3 ml of 95% ethanol and centrifuged at 5,000 x g for 10 minutes. The resulting supernatant (2.1ml) was pipetted and loaded onto a Sephadex LH-20 column (2 x 27 cm) equilibrated in 95% ethanol. The non-tannin polyphenolic compounds were firstly eluted by 95% ethanol followed by the elution of the tannins by 50% aqueous acetone as described by Strumayer and Malin (1975). In all, 50 ethanol fractions and 30 acetone fractions were collected for each of the five cowpea samples. Absorbance per gramme of seed of both the ethanol and acetone eluants were also determined.

Elution profile for each cowpea sample was constructed from the absorbances of the eluants. Peak fractions of the cowpea extracts were pooled together and tannin contents determined in them. Seed coat tannin contents were again determined directly from the extracts without passing them through the Sephadex LH-20 column. Ground seed coats (100 mg) in four replicates were weighed into centrifuge tubes and 1ml of solvent (1% HCl in one experiment or 70% acqueous acetone in another) added to each tube. The tubes were shaken at 5 minutes intervals whilst being held at 30 oC for 30 minutes. The extracts in the tubes were centrifuged at 10,000 x g and the supernatants decanted into 5 ml volumetric flasks. The sediments were re-extracted twice more with 1ml of solvent at a time and these extracts added to the first ones. The extracts in the volumetric flasks were made up to 5 ml by adding more of the solvent. These were filtered through Whatman No. 1 filter paper and concentrated to dryness before being redissolved in 2.5 ml of methanol.

The assay for determining the tannin content was carried out in a water bath at 30 oC following the vanillin-HCl procedure (Burns, 1970; Price et al., 1970). From each set of concentrated pooled fractions, 0.1ml aliquot was pipetted into the test tubes in quadruplicates and 0.9 ml of methanol added to make up to 1.0 ml. Vanillin reagent (5 ml), prepared daily by adding equal volumes of 1% vanellin in methanol and 8% HCl in methanol, was added to the first set of test tubes at 1 minute intervals. To correct for the colour background, 5ml 4% HCl was added to a second set of test tubes at 1 minute intervals after 0.1ml aliquot and 0.9ml methanol had been added and the absorbance read, subtracted from the former reading. The test tubes were incubated in the water bath for a further 20 minutes after which their absorbances were read at 500 nm. The tannin contents in mg catechin equivalent in 1.0 ml of the extracts were then determined from standard curves.

The standard curves were constructed for each of the five cowpea samples separated on the Sephadex LH-20 column. In addition, standard curves were constructed for the other extractions (1% methanolic HCl and 70% aqueous acetone). The catechin reagent used to construct the standard curves was prepared daily by dissolving 25 mg in 25 ml of methanol (i.e., 1.0 mg^-1). Various volume, 0.0, 0.1, 0.2, 0.2 and 0.8 ml in 25 ml of the catechin reagent were pipetted into test tubes and methanol added to them to make up to 1.0 ml. After addition of 5 ml vanillin reagent, the test tubes were left to incubate for 20 minutes in a water bath, after which their absorbances were read at 500 nm. The tannin contents in the eluants of each cowpea cultivar was then determined for each cowpea sample, taking the dilution into consideration.

Determination of seed coat lignin content. Seed coats of the five cowpea samples were manually removed and ground in a hammer mill to pass a 1mm screen. A procedure using aqueous acetone (Haslam, 1966) for the removal of tannins was used. The samples (10-15 mg) for lignin determination were weighed and 2ml of acetone:water (70:30; v:v) were added. The vials were capped, shaken and left for 10 minutes at room temperature (20-22oC) before centrifuging at 500 x g for 5 minutes. The solution was pipetted off and the extraction with acetone:water repeated twice. The samples were finally extracted with acetone and dried at 50oC overnight. Lignin contents were determined directly on the residues.

Lignin was determined by the modified acetyl bromide procedure of Liyama and Wallis (1988) except that 10-15 mg samples were weighed into 4ml brown vials and 2.0 ml of 25% acetyl bromide containing perchloric acid (70%, 0.08 ml) was added. After digestion, the samples were transferred, after dissolving in acetic acid, to 50 ml volumetric flasks containing 2M sodium hydroxide (5 ml) and acetic acid (12 ml). The flasks were made to the mark with acetic acid. Analyses were carried out in duplicate samples of seed coat material.

Relationships between seed storage potential and seed coat chemical analysis. Correlations were calculated between the parameters of seed quality during storage and the seed coat chemical analysis using Pearson’s correlation coefficient.

RESULTS

The initial percentage germinations for the five cultivars were high, over 90% (Tables 1 and 2). Subsequently, the percentage germination decreased progressively in all the cultivars as the ageing period increased in both controlled deterioration and accelerated ageing. The unpigment cultivars showed more reduction in germination than the pigmented cultivars; for example, at the end of six days accelerated ageing, germination percentages of the unpigmented cultivars had dropped from over 92 to 32-44%, whereas during this same period, the pigmented cultivars showed less reduction, with final germination above 80%.

To examine whether the differences in germination between the pigmented and unpigmented cultivars or unaged and aged seeds were reflected in the solute leakage from the seeds, seed leachate conductivity (µamps g^-1) was measured. Solute leakage showed progressive increases as ageing period increased, from the initial 280 µamps g^-1 to 399-605 and 179-233 µamps g^-1 for the unpigmented and the pigmented cultivars, respectively (Table 3). This revealed the reduced ability of cell membrane to retain its solute content when seeds were aged, particularly in the unpigmented cultivars.

Percentages of complete vital staining of unstored seed embryos imbibed in water for 24 hours were generally high (80-100) in all the five cultivars particularly in the pigmented ones (Table 3). However, these percentages dropped sharply to 0% for the unpigmented seeds aged beyond 4 days. The pigmented seeds retained high levels of vital staning until the sixth day when the percentages dropped to 45 and 50% for the two pigmented cultivars. This indicated that the incidence of imbitition damage occurred progressively with increasing storage and this was greater in unpigmented seeds. These results revealed an increasing susceptibility to imbibition damage in aged seeds when imbibed in free water. The increased susceptibility to imbibition damage occurred earlier (2 days during accelerated ageing) in the unpigmented seeds than in the pigmented seeds (4 days during accelerated ageing). This suggests that imbibition damage is associated with reduced germination, increased solute leakage and reduced vital staining.

To determine if the differences in susceptibility to imbibition damage between the unpigmented and the pigmented cultivars, as well as stored and unstored seeds, were associated with any differences in the rate of water uptake, seeds were imbibed in free water before and during storage. The rate of imbibition before storage showed a clear difference between pigmented and unpigmented cultivars (Table 4). The pigmented cultivars initially imbibed water more slowly while unpigmented cultivars imbibed more rapidly. Following various periods of ageing, the pigmented cultivars showed gradual increases in the rate of water uptake and at the end of the six days accelerated ageing, the rate of water uptake for the pigmented cultivars was close to that of the unpigmented cultivars. The rate of water uptake in the unpigmented cultivars was equally rapid before and during storage. Thus, increase in the susceptibility to imbibition damage in the unpigmented seeds might have resulted from decrease in cell membrane integrity but not from increases in the rate of water uptake. However, increase in imbibition damage in the pigmented seeds during storage may have resulted from the increases in rate of water uptake in these cultivars as well as decreases in cell membrane integrity. The accelerated ageing and prolonged storage experiments showed similar changes in germination, imbibition damage, and rate of water uptake. The correlations between germination, leachate conductivity, and complete vital staining of the accelerated ageing versus the six-months storage experiments were highly significant (P< 0.001; Table 5), indicating that accelerated ageing could be used to predict success of long-term storage.

Seed coat chemical analyses were conducted to determine if the differences in physiological reaction between pigmented and unpigmented cultivars of cowpea could be related to chemical compounds in the seed coat. Absorbance per gramme of the ethanol eluants of pigmented seed coat extracts fractionated on Sephadex LH-20 column were 3 times that of the unpigmented cultivars (Table 6). Also the absorbance per gramme of the acetone eluants were about 11-15 times that of seed with unpigmented seed coats.

The tannin contents (mg catechin equivalent g^-1 of the cowpea cultivars) determined in eluants of the Sephadex LH-20 column as well as in the 1% methanolic HC1 and 70% aqueous acetone extracts were extremely low in the white-seeded cultivars (Table 6). The pigmented cultivars showed very high tannin contents, particularly in the brown one (TVX 2724-01F). The lignin content (mg g^-1) of the unpigmented cultivars were about half of the pigmented ones (Table 6). There was a highly significant (P<0.001) correlation between seed coat lignin content and seed coat tannin contents determined in Sephadex LH-20 column (r=0.964), ethanol extracts (r=0.964) and the 70% aqueous acetone extract (r=0.991). The relationships between the seed coat chemical analyses and measurements of factors determining seed storage potential of cowpeas are presented in Table 7. There were negative and highly significant correlations between the seed coat chemical analyses (including absorbances of the eluants from the Sephadex LH-20 column, tannin contents determined from the three extracts and the lignin content), on one hand, and the rate of water uptake as well as the electrical conductivity of seed leachate, on the other. There were positive and highly significant (P<0.01) correlations between the seed coat chemical analyses and percentage vital staining during accelerated ageing and the prolonged simulated tropical storage. The correlation between the chemical analyses and percentage germination during accelerated ageing, prolonged storage and controlled deterioration (except with acetone A₄₀₀ g^-1) were all significant (P<0.01). Thus, tannins, lignin, and other chemical compounds in the seed coats of cowpea have significant influence on the rate of water uptake, imbibition damage (as revealed by vital staining of the cotyledons and seed leachate conductivity) and germination of cowpea during storage.

DISCUSSION

Reduced germination was observed as ageing period progressed, particularly in the unpigmented cultivars (Tables 1 and 2). Similar observations were made by Akinola et al. (1999), and Bingham and Merritt (1999).

Throughout the storage period, the early development of dead tissues on the cotyledons of the unpigmented cultivars was apparent; in the pigmented cultivars, this occurred during the later stages of ageing and reflected on their less rapid deterioration. The decreases in vital staining of the cotyledons of both pigmented and unpigmented seeds during ageing was due to the occurrences of dead tissues and increasing susceptibility to imbibition damage. Powell and Matthews (1978) have suggested that imbibition damage results from the disruption of weak cell membranes due to the inability of this membrane to withstand the rapid inrush of water.

The increase in susceptibility of aged peas to imbibition damage following storage in the warm and humid environment was associated with a reduction in total membrane phospholipids (Powell and Matthews, 1981a) which resulted in a reduction in the total membrane volume and hence a weakening of the membranes. Further evidence of weakened membranes during ageing was reported by Parrish et al. (1982) who showed that the tugor pressure of the cell membrane of soybean seeds subjected to accelerated ageing was significantly reduced upon hydration. This may explain why the seeds of unpigmented cultivars that originally showed high rates of water uptake kept on increasing in susceptibility to imbibition damage (despite maintaining their rates of water uptake) and decreasing in germination as the storage period progressed. This may also explain why the pigmented cultivars also showed increasing evidence of imbibtion damage later during storage. Thus, the susceptibility to imbibition damage in both deteriorated and undeteriorated seeds is related to the physiological condition of the seed and also to the characteristic rate of water uptake which is inherent in unaged seed.

In the seed coat chemical analysis, distinct differences between the pigmented and unpigmented cultivars were observed. The absorbance per gramme of seed of the ethanol and acetone eluants were 3 and 11 - 15 times more in the pigmented seed coats, respectively (Table 6). Tannin contents of the three extracts were 100-1000 times more in the pigmented seed coats. Such high differences in tannin content had also been found in faba bean (Bond, 1975; Mardquardt et al., 1978; Cabrera and Martin, 1989; Cabrera et a., 1989; Bond and Smith, 1989, van Loon et al., 1989; Bos and Jetten, 1989; de Haro et al., 1989; Moneam, 1990) and cowpea (Morrison et al., 1995).

The progressive increases in the rate of water uptake in the pigmented seeds in storage may be explained by the observation of Sousa and Marcos-Filho (1993) who found that in Calopogonium mucunoides, deteriorated seeds imbibed water more quickly and revealed poor vital staining. This was the result of changes in the unstable waxy deposits in the shallow depressions in their seed coats by the modification of the storage environment (warm and humid) as described by Ragus (1987). Also, relative humidities above 74.1% increased the softening rate of seed coats of subterranean clover suggesting that the slow imbibing cultivars might become increasingly permeable during storage in a warm and humid environment (Fairbrother, 1991). In chickpea, the increased rate of imbition following storage resulted from a slight expansion of the seed coat caused by increased seed moisture content (Legesse, 1991) followed by drying back. In cowpea, however, it has been reported that seed coat permeability is a major factor influencing water uptake and adherence of the seed coat to the cotyledons appears to play a minor role (Legesse and Powell, 1992). The distinct differences between the pigmented and unpigmented seeds in terms of quality changes during storage were therefore clearly related to the seed coat chemical compounds.

CONCLUSIONS

The unpigmented cultivars showed more rapid deterioration in terms of reduced germination and greater susceptibility to imbibition damage during controlled deterioration, accelerated ageing and simulated tropical storage. The greater susceptibility of aged seeds to imbibition damage, particularly the unpigmented seeds even when germination percentage had not dropped significantly, supports the hypothesis that membrane deterioration occurs at an early stage of ageing. Cowpea seed coat pigmentation was closely related to the chemical composition. Thus, the chemical compounds collectively influenced the physiological quality of the seed such as germination, imbibition damage and water uptake. This should be an important factor for plant breeders to consider when selecting for new cultivars adapted to stress conditions.

REFERENCES

1. Akinola, J.O., Jji, P.A., Bonidefaiye, S.O. and Olorunju, S.A.S. 1999. Studies on seed germination and seedling emergence in Leucaena leucocephala (Lam) de Wit cv. Peru. Seed Science and Technology 27:123-129.
2. Asiedu, E. A. and Powell. A. A. 1998. Comparison of the storage potential of cultivars of cowpea (Vigna unguiculata) differing in seed coat pimentation. Seed Science and Technology 26:211-221.
3. Association of Official Seed Analyst (AOSA). 1981. Rules for testing seeds. Journal of Seed Technology 62:1-126.
4. Bingham, I.J. and Merritt, G.J. 1999. Effects of seed ageing on early post-germination root extension in maize: a spatial and histological analysis of the growth zone. Seed Science and Technology 27:151-162.
5. Bond, D. A. and Smith, D. B. 1989. Possibilities of reduction of antinutritional factors in grain legume. In: Recent Advances of Research in Antinutritional Factors in Legume Seeds. Proceedings of the first International Workshop on Antinutritional Factors (ANF) in Legume Seeds, Wageningen, The Netherlands. 23-25 November, 1989. Huisman, J., van der Poel, T. F. B. and Liener, I. E. (Eds.), pp. 284-296. Pudoc, Wageningen.
6. Bos, K. and Jetten, J. 1989. Determination of tannins in Faba beans. In: Recent Advances of Research in Antinutritional Factors in Legume Seeds. Proceedings of the first International Workshop on Antinutritional Factors (ANF) in Legume Seeds, Wageningen, The Netherlands, 23-25 November, 1989. Huisman, J., van der Poel, T. F. B. and Liener, I. E. (Eds.), pp. 168-171. Pudoc, Wageningen.
7. Bressani, R. and Elias, L. G. 1979. The nutritional role of polyphenols in beans. In: Polyphenol in Cereals and Legumes. Hulse, J. H. (Ed.), pp. 61-68. The International Development Centre, Ottawa.
8. Burns, R. E. 1970. Method of estimation of tannin in grain sorghum. Agronomy Journal 63: 511-513.
9. Cabrera, J., Lopez-Medina, J. and Martin, A. 1989. The genetics of tannin content in Faba bean (Vicia faba.). In: Recent Advances of Research in Antinutritional Factors in Legume Seeds. Proceedings of the first International Workshop on Antinutritionsal Factors (ANF) in Legume Seeds, Wageningen, The Netherlands, 23-25 November, 1989. Huisman, J., van Poel, T. F. B. and Liener, I. E. (Eds.), pp. 297-300. Pudoc, Wageningen.
10. Cabrera, J. and Martin, A. 1989. The genetics of tannin content and its relationship with flower and testa colours in Vicia faba. Journal of Agricultural Science Cambridge 113:93-98
11. Carmona, A., Seidl, S. D. and Faffe, W. G. 1991. Comparison of extraction methods and assay procedures for the determination of the apparent tannin content of common beans. Journal of the Science of Food and Agriculture 56:291-301.
12. Deakin, J. R. 1974. Association of seed colour with emergence and seed yield of snap beans. Journal of the American Society of Horticultural Science 99:110-114.
13. de Haro, A., Lopez-Medina, J., Cabrera, A. and Martin, A. 1989. Determination of tannins in the seeds of Vicea faba by NIR. In: Recent Advances of Research in Antinutritional Factors in Legume Seeds. Proceedings of the first International Workshop on Anti-nutritionsal Factors (ANF) in Legume Seeds, Wageningen, The Netherlands. 23-25 November, 1989. Huisman, J., van Poel, T. F. B. and Liener, I. E. (Eds.), pp. 172-175. Pudoc, Wageningen.
14. Delouche, J. C. and Baskin, C.C. 1973. Accelerated ageing techniques for predicting the relative storability of seed lots. Seed Science and Technology 1:427-452.
15. Fairbrother, T. E. 1991. Effect of fluctuating temperatures and humidity on the softening rate of hard seed of suberranean clover (Trifolium subterraneum L.). Seed Science and Technnology 19:93-105.
16. Garrido, A., Cabrera, A. and Guerrero, J, E. 1989. Relationship between tannin content and in vitro nutritive value in seeds of 24 strains of Vicia faba. In: Recent Advances of Research in Antinutritional Factors in Legume Seeds. Proceedings of the first International Workshop on Antinutritional Factors (ANF) in Legume Seeds, Wageningen, The Netherlands, 23-25 November 1989. Huisman, J., van der Poel, T. F. B. and Liener, I. E. (Eds.), pp. 172-175. Pudoc, Wageningen
17. Gatel, F. and Gosjean, F. 1990. Composition and nutritive value of peas for pigs: a review of European results. Livestock Production Science 26:155-175.
18. Haslam, E. 1966. Chemistry of Vegetable Tannins. London, Academic Press.
19. Kannenberg, L. W. and Allard, R. W. 1960. An association between pigment and lignin formation in the seed coat of the Lima bean. Crop Science 4:621-622.
20. Knights, E. J. and Mailer, R. J. 1989. Association of seed type and colour with establishment, yield and seed quality in chickpea (Cicer arietinum). Journal of Agricultural Science Cambridge 133:325-330.
21. Legesse, N. 1991. Genotypic comparisons of imbibition in Chickpea (Cicer arietinum L.) and cowpea (Vigna unguiculata (L.) Walp. Ph.D. Thesis, University of Aberdeen.
22. Legesse, N. and Powell, A. A. 1992. Comparisons of water uptake and imbibition damage in eleven cowpea cultivars. Seed Science and Technology 20:173-180.
23. Matthews, S. 1980. Controlled deterioration: a new vigour test for crop seeds. In: Seed Production. Hebblethwaited, P.D. (Ed.), pp. 647-660. London: Butterworths.
24. Marquardt, R. R., Wark, A. T. and Evans, L. E. 1978. Comparative properties of tannin-free and tannin-containing cultivars of Faba beans (Vicia faba). Canadian Journal of Plant Science 58:753-760.
25. Moneam, N. M. 1990. Effects of presoaking on Faba bean enzyme inhibitors and polyphenols after cooking. Journal of Agriculture and Food Chemistry 38:1479-1482.
26. Morrison, I., Asiedu, E. A., Stuchbury, T. and Powell, A. A. 1995. Determination of tannins and lignin in cowpea seed coat. Annals of Botany 76:287-290.
27. Nozzolillo, C., Ricciarke, L. and Lattanzio, V. 1989. Flavonoid constituents of Vicia faba (Fabaceae) in relation to genetic control of their colour. Canadian Journal of Botany 67:1600-1604.
28. Parish, D. J., Leopold, A. C. and Hanna, M. A. 1982. Turgor changes with accelerated ageing of soybean. Crop Science 22:666-669.
29. Price, M. L., Scoyoc, S. V. and Butler, L. G. 1978. A critical evluation of the Vanillin reaction as an assay for tannin in sorghum grain. Journal of Agriculture and Food Chemistry 26:1214-1219.
30. Powell, A. A. and Matthews, S. 1981a. Asso-ciation of phospholipids changes with early stages of seed ageing. Annals of Botany 47:709-712.
31. Powell, A. A. and Matthews, S. 1981b. Evaluation of controlled deterioration, a new vigour test for crop seeds. Seed Science and Technology 9:633-640.
32. Powell, A. A., Oliveira, M de A. and Matthews, S. 1986a. Seed vigour in cultivars of dwarf French bean (Phaseolus vulgaris, L.) in relation to the colour of the testa. Journal of Agricultural Science, Cambridge 106:419-425
33. Powell, A. A., Oliveira, M de A. and Matthews, S. 1986b. The role of imbibition damage in determining the vigour of white and coloured seed lots of dwarf Frech beans (Phaseolus vulgaris).Journal of Experimental Botany 37:716-722.
34. Powell, A. A. and Matthews, S. 1978. Deteriorative changes in pea seeds (Pisum
35. sativum L.) stored in humid or dry conditions. Journal of Experimental Botany 28:225-234.
36. Ragus, L. N. 1987. Role of water absorbing capacity in soybean germination and seedling vigour. Seed Science and Technology 15:285-296.
37. Sausa, F. H.D. and Marcos-Filho, F. 1993. Physiological characteristics associated with water imbibition by Calopogonium mucu-noides Desv. Seed Science and Technology 15:285-296.
38. Strumayer, D. H. and Malin, M. J. 1975. Condensed tannins in grain sorghum: Isolation fractionation, and characterization. Journal of Agriculture and Food Chemistry 23:909-914.
39. TeKrony, D. 1995. Accelerated ageing. In: International Seed Testing Association, Seed Vigour Testing Seminar. van de Venter, H. A. (Ed.), pp. 53-72, International Seed Testing Association, Zurich.
40. van der Poel, A. F. B., Gravendeel, S. and Boer, H. 1991. Effect of different processing methods on tannin content and in vitro protein digestibility. Animal Feed Science and Technology 33:49-58.
41. Van Loon, J. J., van Norel, A. and Dllaert, L. M. W. 1989. Tannin-free Vcia faba and disease resistance: conflicting breeding objectives? In: Recent Advances of Research in Antinutritional Factors in Legume Seeds. Proceedings of the First International Workshop on Antinutritional Factors (ANF) in Legume Seeds, Wageningen, The Netherlands, 23-25 November 1989. Huisman, J., van der Poel, T. F. B. and Liener, I. E. (Eds.), pp. 172-175. Pudoc, Wageningen.
42. Winston, S. W. and Bates, D. H. 1960. Saturated solutions for the control of humidity in biological research. Ecology 41:232-237.