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Short Communications

The influence of impaction and sulphuric acid scarification on electrolyte and carbohydrate leakage in Aspalathus linearis seeds

Kathleen M. Kelly* and J. van Staden UN/CSIR Research Unit for Plant Growth and Development, Department of Botany, University of Natal, P.O. Box 375, Pietermaritzburg, 3200 Republic of South Africa

Accepted 10 February 1988

Dormancy in Aspalathus linearis seeds, imposed by a water­impermeable seed coat, may be broken by impaction (percus­sion) or by means of acid scarification. The latter treatment could cause harmful leakage of seed compounds during germination. Leakage from acid-scarified seeds occurred in two phases, with an apparent period of membrane re-establishment between the two phases. This was observed with respect to both electrolyte and carbohydrate leakage. Impacted seeds exhibited a minimal electrolyte and carbohydrate leakage.

Die onnodige uitloog van verbindings vanuit Aspalathus linearis­sade kan verhoed word deur die keuse van behandeling wat die saadhuid deurdringbaar maak. Sade wat geskud (gestamp) is, het 'n minimum van elektroliet- en koolhidraatuitloging getoon. Uitloging van suurgeskarifiseerde sade het in twee fases geskied, met'n klaarblyklike peri ode van membraanherstel tussen die twee fases. Hierdie verskynsel is waargeneem ten opsigte van beide elektroliet- en koolhidraatuitloging.

Keywords: Impaction, leakage, legume, lens, scarification

*To whom correspondence should be addressed

Dormancy in legumes can be broken by rendering the in­tact seed coat permeable to water. A variety of artificial

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methods can be used to alter the nature of the mature seed coat (Rolston 1978). However, the seed coat protects the embryo from cellular rupture and the consequent unneces­sary leakage of intracellular substances during imbibition (Duke & Kakefuda 1981; Duke et al. 1986). Such unneces-

. sary leakage from the seed may decrease seed viability and seedling vigour (Larson 1968; Matthews & Bradnock 1968). It is therefore desirable to find a treatment that will increase the permeability of the coat without impairing its protective function.

Recently an impaction treatment was recommended for Aspalathus linearis (Burmann) Dahlgren as an alternative to sulphuric acid scarification (Kelly & van Staden 1987a). Sulphuric acid scarification caused uneven thin­ning and the random removal of patches of the seed coat , resulting in rapid hydration and the exposure of the embryo to uneven pressure during imbibition (Kelly & van Staden 1985). Impaction, on the other hand, caused only the mac­rosclereid cells at the lens to become raised resulting in a controlled hydration of the coat and embryo (Kelly & van Staden 1987a). It was decided to determine the extent to which the method of coat treatment affects electrolyte and carbohydrate leakage from the embryo, which could in tum influence seed viability and seedling vigour.

Intact, one-year-old seeds of A. linearis were used for all experiments. For the sulphuric acid scarification 10 re­plicates of 10 seeds were immersed in concentrated sulphu­ric acid for 0, 60, 90, 120 and 180 min. Seeds were then washed in running water for 1 min. For the impaction treat­ment seeds were placed in 50 cm3 flasks, sealed with alumi­nium foil and shaken for 12 h at 250 oscillations per min. Electrolyte leakage was studied by soaking 1,5 g impacted or acid-scarified seeds in 20 cm3 distilled water for 6, 12, 24, 36 and 48 h. The electrolyte conductivity of the seed steep water (20 cm') was then measured using a standard con­ductivity meter. Experiments were carried out at room temperature (± 22°C). After the electrolyte conductivity had been determined this seed steep water was taken to dryness at 35°C, resuspended in 5 cm3 10% iso-propanol, and the sugars were then separated by g.l.c. as outlined by Kelly & van Staden (1987b). Both experiments were repeated twice.

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SEED TREATMENTS

Figure 1 Electrolyte leakage from untreated impermeable seeds (-), impacted seeds (.), seeds acid-scarified for 60 (r!!ll) , 90 (D), 120 (~) and 180 (1Zl) min. Electrolyte conductivity was measured in the seed steep water after 6 (A), 12 (B), 24 (C), 36 (D) and 48 (E) h. Bars represent LSD when p = 0,01 for each time period.

S. Afr. 1. Bot., 1988, 54(4)

The leachate conductivity measured from the impacted seeds was minimal for all the soak times (Figure 1). The level of electrolytes which leaked into the seed steep water of the acid-scarified seeds increased progressively as the period of immersion in the acid increased (Figure 1). This leakage occurred in two phases. The first phase occurred over 12 h (Figure lA & B), followed by the second phase over the following 36 h (Figure lC to IE). This pattern suggests that a degree of corrective control was imposed in all seeds irrespective of the choice of treatment. This is con­sistent with the interpretation that leakage during the initial phases of imbibition is due to passive diffusion during a period of membrane re-establishment (Simon 1974). Although re-establishment was apparently successful here, the second phase of leakage cannot be explained. Electro­lyte leakage was due to extensive removal of the seed coat as a result of acid scarification.

Leakage need not necessarily reflect cellular rupture (Duke et al. 1983) . In this case sulphuric acid probably acted by dehydrating the cells (Duran & Estrella Tortosa 1985), and it would appear that the rapid rehydration of dehydrated cells resulted in cellular rupture (Senaratna & McKersie 1983), as was previously suggested for acid-scari­fied seeds of A. linearis (Kelly & van Staden 1985).

Cellular rupture is also reflected in the extent of car­bohydrate leakage from seeds (Senaratna & McKersie 1983). Carbohydrate analysis of the seed steep water con­firmed that there were two definite phases of leakage with respect to the acid-scarified seeds (Figure 2). The increase in electrolyte conductivity of the seed steep water over 12 h (Figure IB) was accompanied by a 3- or 4-fold increase in

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Figure 2 Amount of sugars detected by GLC in the seed steep water of intact AspalathuS linearis seeds which were impacted (12 h) (A) or acid­scarified (180 min) (B) and then soaked for 6, 12,24 and 48 h. Standards represent concentration of sugars in the crushed seed coat and em­bryo of untreated seeds. Sucrose (e), inositol (0 ), glucose (L. ), ga­lactose (0) , fructose (_) and rhamnose (*). Bars represent LSD atp = 0,01 for each time period.

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the levels of sugars which co-chromatographed with fruc­tose, inositol and sucrose (Figure 2B) . A decrease in these levels was attained after 24 h which could not be maintained and the level of sugars again increased in the seed steep water over 48 h of soaking (Figure 2B). The concentration of sugars identified in the steep water of the impacted seeds was lower than that recorded for the homogenized seed coat and embryo (Figure 2A). Thus the integrity of the seed coat of the impacted seeds was apparently maintained throughout the soaking treatment.

The seed coat need not be entirely intact (i.e . untreated) to prevent unnecessary leakage. Impaction permitted water to enter the seed but the lens simultaneously main­tained a 'control ' resulting in minimal leakage (Kelly & van Staden 1987a).

Continual soaking of seeds in 20 cm3 of distilled water for 6 to 48 h and then measuring electrolyte and carbohydrate leakage may appear to be a severe treatment , and it may explain the resumption of leakage after 24 h. Prolonged soaking could have caused new stress in the seeds and thus further leakage. Yet, impaction proved to be a more suit­able method for breaking dormancy as leakage from the treated seeds was controlled.

Acknowledgements To the CSIR for financial support and the Rooibos Tea In­dustry for the seed.

References DUKE, S.H. & KAKEFUDA, G. 1981. Role of the testa in prevent­

ing cellular rupture during imbibition of seeds. Pl. Physiol. 67: 449--456.

DUKE , S.H ., KAKEFUDA, G . & HARVEY, T.M. 1983. Dif­ferential leakage of intracellular substances from imbibing soybean Glycine max seeds. Pl. Physiol. 72: 919-924.

DUKE, S.H., KAKEFUDA , G. , HENSON, CA., LOEFFLER, N.L. & VAN HULLE, N.M. 1986. Role of the testa epidermis in the leakage of intracellular substances from imbibing soybean seeds and its implications for seedling survival. Physiologia Pl. 68: 625---{j31.

DURAN, 1.M. & ESTRELLA TORTOSA, M. 1985. The effect of mechanical and chemical scarification on germination of charlock (Sinapis arvensis L.) seeds. Seed Sci. & Tech. 13: 155-163.

KELLY, K.M . & VAN STADEN, 1. 1985. The effect of acid scarifi­cation on seed coat structure , germination and seedling vigour of Aspalathus linearis. 1. Pl. Physiol. 121: 37-45.

KELLY, K.M. & VAN STADEN, 1. 1987a. The lens as the site of permeability in the papilionoid seed Aspalathus linearis. 1. Pl . Physiol. 128: 395-404.

KELLY , K.M. & VAN STADEN, 1. 1987b. The transport and me­tabolism of sucrose and acetate in guayule (Parthenium argen­tatum Gray) in summer. 1. Pl. Physiol. 127: 261-270.

LARSON, L.A. 1968. The effect soaking pea seeds with or without seed coats has on seedling growth. Pl. Physiol. 43: 255-259.

MATIHEWS, S. & BRADNOCK, W.T. 1968. Relationship be­tween seed exudation and field emergence in peas and French beans. Hort . Res. 8: 89-93.

ROLSTON, M.P. 1978. Water impermeable seed dormancy. Bot. Rev. 44: 365-396.

SENARATNA , T. & McKERSIE, B.D. 1983. Characterization of solute efflux from dehydration-injured soybeans (Glycine max L. Merv) seeds. Pl. Physiol. 72: 911-914.

SIMON, E.W. 1974. Phospholipids and plant membrane per­meability. New Phytol. 73: 337-420.