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Effect of Acid Scarification on Seed Coat Structure, Germination and Seedling Vigour of Aspalathus linearis
Effect of Acid Scarification on Seed Coat Structure, Germination and Seedling Vigour of Aspalathus linearis K. M.
KELLY
and]. VAN STADEN
UN/CSIR Research Unit for Plant Growth and Development, Department of Botany, University of Natal, Pietermaritzburg, 3200, Republic of South Africa Received March 7,1985 . Accepted May 14,1985 Summary Sulphuric acid scarification of Aspalathus linearis seed reduced their impermeability and increased their germination by 100%. Treatments of 120 minutes altered the seed coat structure, extensively damaging cuticle, macrosclereid, osteosclereid, hilar, strophiolar and cotyledon layers. Seedling vigour was adversely affected by scarification. This resulted in a decrease in seedling fresh mass and an increase in seedling abnormalities which increased their mortality. Key words: Aspalathus linearis, germination, scarification, seed coat, seedling vigour.
Introduction The presence of an impermeable seed coat in the Leguminosae is well documented (Hutton and Porter, 1937; Rolston, 1978; Werker, 1980/81). Seed germination usually occurs once the seed coat has been partially removed allowing for the entry of water and gases. In most instances agriculturists employ a scarification treatment to ensure a high germination percentage. Both mechanical and chemical scarification treatments are successful in that they reduce impermeability. Frequently however, these treatments damage seeds and seedlings (Nelson, 1924, 1927; Brown and Booysen, 1969; Ibrahim, 1982). Aspalathus linearis is one of few indigenous South African plants which is presently commercially exploited. This legume which is used for tea production is cultivated under semi-arid conditions. A problem currently besetting the industry is that seedlings obtained following acid scarification, show low vigour, and consequently a high rate of mortality. An additional problem is that the lifespan of commercial plants is steadily decreasing. This makes crop assessment difficult and increases production costs. The present investigation was therefore initiated to critically assess the effect of acid scarification on germination and seedling viability of this species. The ultimate aim being to recommend treatments which do not result in a loss of seedlings or a reduction in their viability and to better understand the role of the legume seed coat.
Materials and Methods Mature seeds of Aspalathus linearis {Burmann} R. M. T. Dahlgren were collected in their natural habitat in the South-Western Cape during 1984. The seed sample contained seeds of
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four colours viz. orange, brown, white and green. Seeds were soned under a dissecting microscope and found to contain 96 % orange and brown seeds which were impermeable, 3.4 % white permeable seeds, and 0.6 % green non-viable seeds, which were discarded. Germination studies were performed on moist filter paper in petri dishes at 20°C. Throughout this investigation ten replicates of ten seeds each were used per treatment. The criterion for germination was the protrusion of the radicle from the covering structures. The inhibition pattern of control and acid scarified seeds was investigated. Acid scarification was achieved by immersing seeds in concentrated sulphuric acid for 0, 30, 60 and 120 minutes. Following this treatment the seeds were washed in running tap water for 3 minutes. Seeds, 100 per treatment, were then weighed individually and incubated in repli dishes. The control and scarified seeds were subsequently weighed at regular intervals for a period of 60 hours to establish their patterns of water uptake. Germination was also recorded. The extent of coat damage as a result of acid treatment was determined by means of light and scanning electron microscopy. For light microscopy white, permeable seeds and brown and orange acid scarified seeds were imbibed in 0.1 % fast green for 3 hours, briefly rinsed to remove excess dye, bisected longitudinally and transversely, and then observed under a dissecting microscope to establish dye penetration. For scanning electron microscopy seeds were fractured in liquid nitrogen and mounted on stubs. Seed coat surfaces were coated with gold palladium in a Polaron E 5000 sputter coater for 4 to 6 minutes. All specimens were viewed with a Jeol JEM T200 microscope. Seedling vigour and development was recorded ten days after scarification and the commencement of incubation. The seedlings were blotted dry and their fresh mass recorded. Observations were made with respect to their morphological appearance.
Fig. 1: Scanning electron micrograph showing the testa topography of a white, unimbibed permeable seed of Aspalathus linearis. Raised areas (arrow) demarcate «bundles or groups» of 7 - 8 macrosclereid cells. Brown and orange seeds had similar surface sculpturing. Insen is a longitudinal view of the macrosclereid layer of an unimbibed white seed (1.5 cm = 200 pm). Fig. 2: As in 1, except that the permeable white seed had been imbibed for 3 hours. The «bundles or groups» of macrosclereids (M) are now clearly visible. Fig. 3: A scanning electron micrograph of an impermeable orange seed after 120 minute acid scarification. Most testa damage was due to the removal of «bundles» of macrosclereids, one such «bundle» is demarcated by the two arrows (brown seeds behaved similarly). Insen is a longitudinal view of an impermeable orange seed prior to scarification (1 em = 100 pm). Fig. 4: Severe acid damage to an impermeable seed after 120 minute acid treatment. Macrosclereid (M) and osteosclereid (0) layers were separated. Cracks (C) appeared in the macrosclereid layer where «bundles» of macrosclereids were removed. Fig. 5: As for 4, except that the macrosclereid layers (M) and osteosclereids (0) have been panly removed revealing the endosperm (E) layer. Fig. 6: Electron micrograph of an osteosclereid after acid removal of the macrosclereids. The previous attachment of macrosclereids to the osteosclereid is visible (arrow). The appearance of fibrils (F) or strands indicates the severity of the acid treatment. Fig. 7: The effect of 120 minute acid scarification on the strophiole. In addition to cracks (C) appearing in the macrosclereid layer, the macrosclereids became raised and fractionated. Fig. 8: Acid scarification (120 minute treatment) corroded the hilar rim (HR), and completely removed the tracheid bar (TB).
J. Plant Physiol. Vol.
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Fig. 9: The effect of acid scarification on the rate of water uptake of impermeable and permeable
A. linearis seeds at 18°C. Scarification treatments in concentrated sulphuric acid were carried out for 30 minutes (f';--f';), 60 minutes ("'--"'), and 120 minutes (0--0). White permeable seeds ( . - - . ) served as a control. Bars indicate the 95 % confidence limits. The arrows indicate the termination of the experiment at germination.
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Results
Aspalathus linearis produces a typical papilionoid seed (Corner, 1951; Gunn, 1981). The embryo is enclosed by a cuticle, sub-cuticle, macrosclereid, osteosclereid (or hour glass cells) and endosperm layers. Irrespective of colour the testa topography of all unimbibed seeds is similar {Fig. 1). When white permeable seeds imbibe, a marked change which is not observed in brown and orange seeds, becomes apparent. It would appear as though the macrosclereids are pushed together in «groups» or «bundles» {Fig. 2). Following 120 minutes acid scarification the topography of the orange and brown impermeable seeds resembled that of the white permeable seeds (Fig. 3). It is in these areas of weakness, between the «bundles» of macrosclereids, that the sulphuric acid damage is determined by the degree of seed impermeability and the length of treatment. In all treatments the cuticle and sub-cuticle were initially removed. After 120 minutes of acid treatment large cracks appeared in the macrosclereid layer (Fig. 4) which was torn from the underlying osteosclereids (Fig. 5), which were apparentlyattached to the macrosclereid fibrils or strands (Fig. 6). Severe acid damage was also observed in the strophiolar (Fig. 7) and hilar regions (Fig. 8) of the impermeable seeds. In the hilar region, which is closely apposed to the radicle, there was corrosion of the hilar rim and complete removal of the tracheid bar. Untreated brown and orange seeds did not imbibe. Following 30 or 60 minutes scarification, seeds imbibed at a similar rate and extent as the permeable white seeds (Fig. 9). Extended scarification accelerated water uptake for the first 12 hours. The removal of the seed coat in random areas allowed the fast green dye to penetrate Table 1: The effect of acid scarification upon the germination of impermeable A. lineans seeds. The experiment was terminated 60 hours after commencement of incubation at 18°C. Scarification time (minutes)
0
30
60
90
120
Percentage germination
0
7
77
87
100
Overall variation was found to be significant at 95 % confidence level. Multiple t-tests were conducted to determine within treatment variation. All treatments were found to be significantly different. L.S.D. = 2.5.
rapidly. The normal pattern of water uptake was such that the fast green dye after 3 hours imbibition had penetrated the palisade layer and no dye was observed in the embryos. Following the 120 minute acid treatment, the dye had, after 3 hours penetrated the palisade layer and concentrated in the embryo or radicle depending on site of entry. All seeds thus treated, became permeable and germinated within 60 hours (Table 1). Shorter periods of scarification were less successful. Although acid scarification was beneficial with respect to final germination, seedling vigour and morphology were adversely affected. All acid treatments, when
J. plant Plrysiol. Vol.
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SCARIFICATION TIME (MINUTES) Fig. 10: The effect of acid scarification upon seedling fresh mass and seedling vigour ten days after germination. The white seeds served as a control (C). Overall variation was found to be significant between control and treated seedlings at 95 % confidence level. Multiple t-tests indicated that the 120 minute treatment had a significantly deleterious effect on seedling fresh mass. Bar represents the L.S.D. The percentage of abnormal seedlings was calculated as a percentage of the total number of seedlings obtained for each treatment and is represented by the figure found in each bar for the five treatments. For this treatment the L.S.D. was 0.65.
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compared to the control (white permeable seeds), resulted in a decrease in fresh seedling mass and an increase in the number of abnormal seedlings (Fig. 10). Seedling abnormalities included a} a complete break between cotyledon and radicle, b} a stunted radicle due to root tip damage or the development of a shallow secondary root system and, c} broken or malformed cotyledons. Discussion The seed coat structure of Aspalathus linearis is typical to that described for other legume species (Corner, 1951; Gunn, 1981). Within each seed batch approximately 96 % of the seeds were brown or orange in colour and impermeable to water. Most of the remaining 4 % were white and readily imbibed and germinated when incubated under favourable conditions. These seeds were more sensitive to acid treatment. Seed colour was a reliable indicator of permeability and susceptibility to acid which imposed a polymorphism on each seed batch (Farmer et aI., 1982). Germination did not improve following a 30 minute acid treatment, which resulted in the partial removal of the cuticular layer of the impermeable seed coats. The presence of a cuticle is therefore not responsible for the impermeable condition. As in other work (Graaff and Van Staden, 1983), the integrity of the macrosclereid layer is thought to be the main cause of the impermeability. The testa topography of the unimbibed seed is composed of ridges and depressions. In the imbibed white seeds, these ridges appear to demarcate the edges of «bundles» of seven or eight macrosclereids, which separate to reveal fissures upon imbibition. If impermeability has arisen as Egley et ai. (1983) proposed, by the conversion of soluble phenolics to insoluble lignin polymers, then these fissures may be the result of unsuccessful (in white permeable seeds) or incomplete (in brown or orange seeds) chemical bonding between the macrosclereids. The testa topography following scarification was altered to resemble that of the naturally imbibing white seed. This would suggest that the acid penetrated the weaker sites on the seed coat and that the degree of attachment be it physical or chemical, of the macrosclereids is very important. All acid treatments altered the macrosclereid layer and improved germination. However, severe seedling damage and a resultant loss in seedling vigour was experienced with prolonged acid treatment. Aspalathus linearis seeds are commonly scarified for up to 4 hours, but it is evident that after 2 hours severe damage to the seed coat, not evident to the naked eye, has been incurred by the seed. In white seeds, the flow of water into the embryos was slow, subsequent seedling growth was normal. Shorter periods of acid immersion (30 to 60 minutes) brought about similar results. Sulphuric acid is known to be a powerful dehydrating and corrosive agent. Corrosion of the weaker regions of the testa between the macroscIereid «bundles», created fissures allowing for the movement of the acid into the embryo. The appearance of «strands» or «fibrils» on the osteoscIereids, following 120 minute acid treatment, is indicative of the destructive power of the acid. As the exposure pe-
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K. M. KELLy and]. VAN STADEN
riod to acid increased, the corrosive action of the acid brought about disruption of the covering structures. Water uptake was completed within 12 hours, in the seeds. Imbibition was rapid and water penetration random. The rapid influx of water combined with the initial dehydration power of the acid, probably were the main causes of subsequent embryo and seedling damage. The water potential in damaged areas is known to increase (Hsaio et al., 1983) which would expose the cotyledons and radicles to uneven pressure. Imbibition under natural conditions would take place at a slower rate against the soil water potential (Perry and Harrison, 1970). It is for this reason that seedling damage in the field is not as common as in the laboratory where use is made of various scarification treatments (Nelson, 1927; Prakobboon, 1982). The most severe seeding damage which occurred as a result of sulphuric acid scarification were abnormalities with respect to radicle and root development. These were similar to the dehydration injuries described by Senaratna and McKersie (1983). The environmental pressures in a semi-arid environment require the production of a substantial root system and an adequate supply of reserves for successful seedling establishment (Tadmor and Cohen, 1968). Normally A. linearis produces a 2m long tap root which is four times the length of the shoot after one month of growth. This enables the seedling to reach a water supply before the seed beds dry out. It is therefore obvious that scarification treatments resulting in the development of shallow root systems, and the breaking of the cotyledons, which alters the food reserve supply of the seedlings, will severely restrict the survival of seedlings in the field. In order to place the Aspalathus tea industry on a better footing it is essential that a means be found to bring about scarification without extensively damaging the macrosclereid layer. This layer, will then, by maintaining equal hydration of tissues during imbibition, protect the embryo from damage and appears to playa dominant role in the maintenance of seedling vigour. Acknowledgements The financial support of the C.S.I.R., Pretoria. The Electron Microscope Unit, Pietermaritzburg, for their help and the Rooibos Tea Industry, Clanwilliam, for the provision of seed.
References BROWN, N. A. C. and P. DE V. BOOYSEN: Seed coat impermeability in several Acacia species. Agroplantae 1, 51-60 (1969). CORNER, E. J. H.: The Leguminous seed. Phytomorphology 1, 117 -121 (1951). EGLEY, G. H., R. N. PAUL, K. C. VAUGHN, and S. O. DUKE: Role of peroxidase in the development of water-impermeable seed coats in Sida spinosa L. Planta 157, 224-232 (1983). FARMER, R. E., G. C. LOCKLEY, and M. CUNNINGHAM: Germination patterns of the sumacs, Rhus glabra and Rhus coppolina: effects of scarification time, temperature and genotype. Seed Sci. Tech. 10,223-231 (1982). GRAAFF, J. L. and J. VAN STADEN: Seed coat structure of Sesbania species. Z. Pflanzenphysiol.
111,293-299 (1983).
GUNN, C. R.: Seeds of Leguminosae. In: POLHILL, R. M. and P. H. RAVEN (eds.): Advances in Legume Systematics, Vol. 11,913-925. Royal Botanic Gardens, Kew, 1981.
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HSAIO, A.I., G.I. McINTYRE, andJ. A. HANES: Seed dormancy in Avenafatua. I. Induction of germination by mechanical injury. Bot. Gaz. 144, 217 -222 (1983). HUTrON, M. E. G. and R. H. PORTER: Seed impermeability and viability of native and introduced species of Leguminosae. Iowa State J. Res. 12, 5-24 (1937). IBRAHIM, A. E. S.: The influence of mechanical damage to the seed coat on the dormancy of groundnut seeds. Ann. Bot. 50, 563 -566 (1982). NELSON, A.: Hard seeds and broken seedlings in red clover. Bot. Soc. Edinburgh Trans. 29, 6668 (1924). - Hard seeds and broken seedlings in red clover. III. Soil effects. Bot. Soc. Edinburgh Trans. 29,402-407 (1927). PERRY, D. A. and J. G. HAIuuSON: The deleterious effect of water and low temperature on germination of pea seed. J. Exp. Bot. 21, 504-512 (1970). PRAKOBBOON, N.: A study of abnormal seedling development in soybean as affected by threshing injury. Seed Sci. Tech. 10, 495-500 (1982). ROLSTON, M. P.: Water impermeable seed dormancy. Bot. Rev. 44, 363-396 (1978). SENARATNA, T. and B. D. McKERSIE: Dehydration injury in germinating soybean (Glycine max L. Merr.) seeds. Plant Physiol. 72, 620-624 (1983). TADMOR, N. H. and Y. COHEN: Root elongation in the pre-emergence stage of Mediterranean grasses and legumes. Crop Sci. 8, 416-419 (1968). WERKER, E.: Seed dormancy as explained by the anatomy of embryo envelopes. Isr. J. Bot. 29, 22-44 (1980/81).
J. Plant Physiol.
Vol. 121. pp. 37-45 (1985)
KELLY
and]. VAN STADEN
UN/CSIR Research Unit for Plant Growth and Development, Department of Botany, University of Natal, Pietermaritzburg, 3200, Republic of South Africa Received March 7,1985 . Accepted May 14,1985 Summary Sulphuric acid scarification of Aspalathus linearis seed reduced their impermeability and increased their germination by 100%. Treatments of 120 minutes altered the seed coat structure, extensively damaging cuticle, macrosclereid, osteosclereid, hilar, strophiolar and cotyledon layers. Seedling vigour was adversely affected by scarification. This resulted in a decrease in seedling fresh mass and an increase in seedling abnormalities which increased their mortality. Key words: Aspalathus linearis, germination, scarification, seed coat, seedling vigour.
Introduction The presence of an impermeable seed coat in the Leguminosae is well documented (Hutton and Porter, 1937; Rolston, 1978; Werker, 1980/81). Seed germination usually occurs once the seed coat has been partially removed allowing for the entry of water and gases. In most instances agriculturists employ a scarification treatment to ensure a high germination percentage. Both mechanical and chemical scarification treatments are successful in that they reduce impermeability. Frequently however, these treatments damage seeds and seedlings (Nelson, 1924, 1927; Brown and Booysen, 1969; Ibrahim, 1982). Aspalathus linearis is one of few indigenous South African plants which is presently commercially exploited. This legume which is used for tea production is cultivated under semi-arid conditions. A problem currently besetting the industry is that seedlings obtained following acid scarification, show low vigour, and consequently a high rate of mortality. An additional problem is that the lifespan of commercial plants is steadily decreasing. This makes crop assessment difficult and increases production costs. The present investigation was therefore initiated to critically assess the effect of acid scarification on germination and seedling viability of this species. The ultimate aim being to recommend treatments which do not result in a loss of seedlings or a reduction in their viability and to better understand the role of the legume seed coat.
Materials and Methods Mature seeds of Aspalathus linearis {Burmann} R. M. T. Dahlgren were collected in their natural habitat in the South-Western Cape during 1984. The seed sample contained seeds of
J. Plant Physiol.
Vol. 121. pp. 37-45 (1985)
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J Plant P/rysiol. Vol.
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four colours viz. orange, brown, white and green. Seeds were soned under a dissecting microscope and found to contain 96 % orange and brown seeds which were impermeable, 3.4 % white permeable seeds, and 0.6 % green non-viable seeds, which were discarded. Germination studies were performed on moist filter paper in petri dishes at 20°C. Throughout this investigation ten replicates of ten seeds each were used per treatment. The criterion for germination was the protrusion of the radicle from the covering structures. The inhibition pattern of control and acid scarified seeds was investigated. Acid scarification was achieved by immersing seeds in concentrated sulphuric acid for 0, 30, 60 and 120 minutes. Following this treatment the seeds were washed in running tap water for 3 minutes. Seeds, 100 per treatment, were then weighed individually and incubated in repli dishes. The control and scarified seeds were subsequently weighed at regular intervals for a period of 60 hours to establish their patterns of water uptake. Germination was also recorded. The extent of coat damage as a result of acid treatment was determined by means of light and scanning electron microscopy. For light microscopy white, permeable seeds and brown and orange acid scarified seeds were imbibed in 0.1 % fast green for 3 hours, briefly rinsed to remove excess dye, bisected longitudinally and transversely, and then observed under a dissecting microscope to establish dye penetration. For scanning electron microscopy seeds were fractured in liquid nitrogen and mounted on stubs. Seed coat surfaces were coated with gold palladium in a Polaron E 5000 sputter coater for 4 to 6 minutes. All specimens were viewed with a Jeol JEM T200 microscope. Seedling vigour and development was recorded ten days after scarification and the commencement of incubation. The seedlings were blotted dry and their fresh mass recorded. Observations were made with respect to their morphological appearance.
Fig. 1: Scanning electron micrograph showing the testa topography of a white, unimbibed permeable seed of Aspalathus linearis. Raised areas (arrow) demarcate «bundles or groups» of 7 - 8 macrosclereid cells. Brown and orange seeds had similar surface sculpturing. Insen is a longitudinal view of the macrosclereid layer of an unimbibed white seed (1.5 cm = 200 pm). Fig. 2: As in 1, except that the permeable white seed had been imbibed for 3 hours. The «bundles or groups» of macrosclereids (M) are now clearly visible. Fig. 3: A scanning electron micrograph of an impermeable orange seed after 120 minute acid scarification. Most testa damage was due to the removal of «bundles» of macrosclereids, one such «bundle» is demarcated by the two arrows (brown seeds behaved similarly). Insen is a longitudinal view of an impermeable orange seed prior to scarification (1 em = 100 pm). Fig. 4: Severe acid damage to an impermeable seed after 120 minute acid treatment. Macrosclereid (M) and osteosclereid (0) layers were separated. Cracks (C) appeared in the macrosclereid layer where «bundles» of macrosclereids were removed. Fig. 5: As for 4, except that the macrosclereid layers (M) and osteosclereids (0) have been panly removed revealing the endosperm (E) layer. Fig. 6: Electron micrograph of an osteosclereid after acid removal of the macrosclereids. The previous attachment of macrosclereids to the osteosclereid is visible (arrow). The appearance of fibrils (F) or strands indicates the severity of the acid treatment. Fig. 7: The effect of 120 minute acid scarification on the strophiole. In addition to cracks (C) appearing in the macrosclereid layer, the macrosclereids became raised and fractionated. Fig. 8: Acid scarification (120 minute treatment) corroded the hilar rim (HR), and completely removed the tracheid bar (TB).
J. Plant Physiol. Vol.
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K. M. KELLY and J.
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Fig. 9: The effect of acid scarification on the rate of water uptake of impermeable and permeable
A. linearis seeds at 18°C. Scarification treatments in concentrated sulphuric acid were carried out for 30 minutes (f';--f';), 60 minutes ("'--"'), and 120 minutes (0--0). White permeable seeds ( . - - . ) served as a control. Bars indicate the 95 % confidence limits. The arrows indicate the termination of the experiment at germination.
! Plant Physiol. Vol.
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Results
Aspalathus linearis produces a typical papilionoid seed (Corner, 1951; Gunn, 1981). The embryo is enclosed by a cuticle, sub-cuticle, macrosclereid, osteosclereid (or hour glass cells) and endosperm layers. Irrespective of colour the testa topography of all unimbibed seeds is similar {Fig. 1). When white permeable seeds imbibe, a marked change which is not observed in brown and orange seeds, becomes apparent. It would appear as though the macrosclereids are pushed together in «groups» or «bundles» {Fig. 2). Following 120 minutes acid scarification the topography of the orange and brown impermeable seeds resembled that of the white permeable seeds (Fig. 3). It is in these areas of weakness, between the «bundles» of macrosclereids, that the sulphuric acid damage is determined by the degree of seed impermeability and the length of treatment. In all treatments the cuticle and sub-cuticle were initially removed. After 120 minutes of acid treatment large cracks appeared in the macrosclereid layer (Fig. 4) which was torn from the underlying osteosclereids (Fig. 5), which were apparentlyattached to the macrosclereid fibrils or strands (Fig. 6). Severe acid damage was also observed in the strophiolar (Fig. 7) and hilar regions (Fig. 8) of the impermeable seeds. In the hilar region, which is closely apposed to the radicle, there was corrosion of the hilar rim and complete removal of the tracheid bar. Untreated brown and orange seeds did not imbibe. Following 30 or 60 minutes scarification, seeds imbibed at a similar rate and extent as the permeable white seeds (Fig. 9). Extended scarification accelerated water uptake for the first 12 hours. The removal of the seed coat in random areas allowed the fast green dye to penetrate Table 1: The effect of acid scarification upon the germination of impermeable A. lineans seeds. The experiment was terminated 60 hours after commencement of incubation at 18°C. Scarification time (minutes)
0
30
60
90
120
Percentage germination
0
7
77
87
100
Overall variation was found to be significant at 95 % confidence level. Multiple t-tests were conducted to determine within treatment variation. All treatments were found to be significantly different. L.S.D. = 2.5.
rapidly. The normal pattern of water uptake was such that the fast green dye after 3 hours imbibition had penetrated the palisade layer and no dye was observed in the embryos. Following the 120 minute acid treatment, the dye had, after 3 hours penetrated the palisade layer and concentrated in the embryo or radicle depending on site of entry. All seeds thus treated, became permeable and germinated within 60 hours (Table 1). Shorter periods of scarification were less successful. Although acid scarification was beneficial with respect to final germination, seedling vigour and morphology were adversely affected. All acid treatments, when
J. plant Plrysiol. Vol.
121. pp. 37-45 (1985)
42
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SCARIFICATION TIME (MINUTES) Fig. 10: The effect of acid scarification upon seedling fresh mass and seedling vigour ten days after germination. The white seeds served as a control (C). Overall variation was found to be significant between control and treated seedlings at 95 % confidence level. Multiple t-tests indicated that the 120 minute treatment had a significantly deleterious effect on seedling fresh mass. Bar represents the L.S.D. The percentage of abnormal seedlings was calculated as a percentage of the total number of seedlings obtained for each treatment and is represented by the figure found in each bar for the five treatments. For this treatment the L.S.D. was 0.65.
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compared to the control (white permeable seeds), resulted in a decrease in fresh seedling mass and an increase in the number of abnormal seedlings (Fig. 10). Seedling abnormalities included a} a complete break between cotyledon and radicle, b} a stunted radicle due to root tip damage or the development of a shallow secondary root system and, c} broken or malformed cotyledons. Discussion The seed coat structure of Aspalathus linearis is typical to that described for other legume species (Corner, 1951; Gunn, 1981). Within each seed batch approximately 96 % of the seeds were brown or orange in colour and impermeable to water. Most of the remaining 4 % were white and readily imbibed and germinated when incubated under favourable conditions. These seeds were more sensitive to acid treatment. Seed colour was a reliable indicator of permeability and susceptibility to acid which imposed a polymorphism on each seed batch (Farmer et aI., 1982). Germination did not improve following a 30 minute acid treatment, which resulted in the partial removal of the cuticular layer of the impermeable seed coats. The presence of a cuticle is therefore not responsible for the impermeable condition. As in other work (Graaff and Van Staden, 1983), the integrity of the macrosclereid layer is thought to be the main cause of the impermeability. The testa topography of the unimbibed seed is composed of ridges and depressions. In the imbibed white seeds, these ridges appear to demarcate the edges of «bundles» of seven or eight macrosclereids, which separate to reveal fissures upon imbibition. If impermeability has arisen as Egley et ai. (1983) proposed, by the conversion of soluble phenolics to insoluble lignin polymers, then these fissures may be the result of unsuccessful (in white permeable seeds) or incomplete (in brown or orange seeds) chemical bonding between the macrosclereids. The testa topography following scarification was altered to resemble that of the naturally imbibing white seed. This would suggest that the acid penetrated the weaker sites on the seed coat and that the degree of attachment be it physical or chemical, of the macrosclereids is very important. All acid treatments altered the macrosclereid layer and improved germination. However, severe seedling damage and a resultant loss in seedling vigour was experienced with prolonged acid treatment. Aspalathus linearis seeds are commonly scarified for up to 4 hours, but it is evident that after 2 hours severe damage to the seed coat, not evident to the naked eye, has been incurred by the seed. In white seeds, the flow of water into the embryos was slow, subsequent seedling growth was normal. Shorter periods of acid immersion (30 to 60 minutes) brought about similar results. Sulphuric acid is known to be a powerful dehydrating and corrosive agent. Corrosion of the weaker regions of the testa between the macroscIereid «bundles», created fissures allowing for the movement of the acid into the embryo. The appearance of «strands» or «fibrils» on the osteoscIereids, following 120 minute acid treatment, is indicative of the destructive power of the acid. As the exposure pe-
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K. M. KELLy and]. VAN STADEN
riod to acid increased, the corrosive action of the acid brought about disruption of the covering structures. Water uptake was completed within 12 hours, in the seeds. Imbibition was rapid and water penetration random. The rapid influx of water combined with the initial dehydration power of the acid, probably were the main causes of subsequent embryo and seedling damage. The water potential in damaged areas is known to increase (Hsaio et al., 1983) which would expose the cotyledons and radicles to uneven pressure. Imbibition under natural conditions would take place at a slower rate against the soil water potential (Perry and Harrison, 1970). It is for this reason that seedling damage in the field is not as common as in the laboratory where use is made of various scarification treatments (Nelson, 1927; Prakobboon, 1982). The most severe seeding damage which occurred as a result of sulphuric acid scarification were abnormalities with respect to radicle and root development. These were similar to the dehydration injuries described by Senaratna and McKersie (1983). The environmental pressures in a semi-arid environment require the production of a substantial root system and an adequate supply of reserves for successful seedling establishment (Tadmor and Cohen, 1968). Normally A. linearis produces a 2m long tap root which is four times the length of the shoot after one month of growth. This enables the seedling to reach a water supply before the seed beds dry out. It is therefore obvious that scarification treatments resulting in the development of shallow root systems, and the breaking of the cotyledons, which alters the food reserve supply of the seedlings, will severely restrict the survival of seedlings in the field. In order to place the Aspalathus tea industry on a better footing it is essential that a means be found to bring about scarification without extensively damaging the macrosclereid layer. This layer, will then, by maintaining equal hydration of tissues during imbibition, protect the embryo from damage and appears to playa dominant role in the maintenance of seedling vigour. Acknowledgements The financial support of the C.S.I.R., Pretoria. The Electron Microscope Unit, Pietermaritzburg, for their help and the Rooibos Tea Industry, Clanwilliam, for the provision of seed.
References BROWN, N. A. C. and P. DE V. BOOYSEN: Seed coat impermeability in several Acacia species. Agroplantae 1, 51-60 (1969). CORNER, E. J. H.: The Leguminous seed. Phytomorphology 1, 117 -121 (1951). EGLEY, G. H., R. N. PAUL, K. C. VAUGHN, and S. O. DUKE: Role of peroxidase in the development of water-impermeable seed coats in Sida spinosa L. Planta 157, 224-232 (1983). FARMER, R. E., G. C. LOCKLEY, and M. CUNNINGHAM: Germination patterns of the sumacs, Rhus glabra and Rhus coppolina: effects of scarification time, temperature and genotype. Seed Sci. Tech. 10,223-231 (1982). GRAAFF, J. L. and J. VAN STADEN: Seed coat structure of Sesbania species. Z. Pflanzenphysiol.
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GUNN, C. R.: Seeds of Leguminosae. In: POLHILL, R. M. and P. H. RAVEN (eds.): Advances in Legume Systematics, Vol. 11,913-925. Royal Botanic Gardens, Kew, 1981.
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HSAIO, A.I., G.I. McINTYRE, andJ. A. HANES: Seed dormancy in Avenafatua. I. Induction of germination by mechanical injury. Bot. Gaz. 144, 217 -222 (1983). HUTrON, M. E. G. and R. H. PORTER: Seed impermeability and viability of native and introduced species of Leguminosae. Iowa State J. Res. 12, 5-24 (1937). IBRAHIM, A. E. S.: The influence of mechanical damage to the seed coat on the dormancy of groundnut seeds. Ann. Bot. 50, 563 -566 (1982). NELSON, A.: Hard seeds and broken seedlings in red clover. Bot. Soc. Edinburgh Trans. 29, 6668 (1924). - Hard seeds and broken seedlings in red clover. III. Soil effects. Bot. Soc. Edinburgh Trans. 29,402-407 (1927). PERRY, D. A. and J. G. HAIuuSON: The deleterious effect of water and low temperature on germination of pea seed. J. Exp. Bot. 21, 504-512 (1970). PRAKOBBOON, N.: A study of abnormal seedling development in soybean as affected by threshing injury. Seed Sci. Tech. 10, 495-500 (1982). ROLSTON, M. P.: Water impermeable seed dormancy. Bot. Rev. 44, 363-396 (1978). SENARATNA, T. and B. D. McKERSIE: Dehydration injury in germinating soybean (Glycine max L. Merr.) seeds. Plant Physiol. 72, 620-624 (1983). TADMOR, N. H. and Y. COHEN: Root elongation in the pre-emergence stage of Mediterranean grasses and legumes. Crop Sci. 8, 416-419 (1968). WERKER, E.: Seed dormancy as explained by the anatomy of embryo envelopes. Isr. J. Bot. 29, 22-44 (1980/81).
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