Water deficit in relation to leaf and stem anatomy of Eucalyptus camaldulensis Dehn. shoots cultivated in vitro

Efeitos da deficiência hídrica induzida in vitro e a anatomia de folha e caule de gemas de Eucalyptus camaldulensis Dehn.

Water deficit in relation to leaf and stem anatomy of Eucalyptus camaldulensis Dehn. shoots cultivated in vitro

Gustavo Maia Souza^1*; Antonio Natal Gonçalves¹; Marcílio de Almeida²

¹Depto. de Ciências Florestais - ESALQ/USP , C.P. 09 - CEP: 13418-900 - Piracicaba, SP.

²Depto. de Ciências Biológicas - ESALQ/USP.

*e-mail: [e-mail]

ABSTRACT: Shoots of E. camaldulensis seedlings from three distinct provenances were submitted to three different levels of in vitro water deficit induced by the addition of sorbitol in the growth media. Transversal sections from the leaf blades and stems were anatomically analysed using the historesin inclusion technique. Shoots from the different provenances presented distinct responses to the treatments. The Gilbert River provenance was the most sensible. The basic alterations found on the anatomical structure in response to the water deficit were: cell collapse, late tissue differentiation, vascular bundle and epidermis disorganization, and alterations on the mesophyll and epidermis thickness.

Key words:Eucalyptus camaldulensis, water deficit, anatomy, tissue culture

Efeitos da deficiência hídrica induzida in vitro e a anatomia de folha e caule de gemas de Eucalyptus camaldulensis Dehn.

RESUMO: Três procedências distintas de E. camaldulensis foram submetidas à diferentes níveis de deficiência hídrica in vitro induzida através da adição de sorbitol em meio de cultura. Cortes transversais de limbos foliares e caules foram analisados anatomicamente através da técnica de emblocamento em historesina. As procedências apresentaram respostas diferenciadas, sendo Gilbert River a mais sensível. As alterações básicas da estrutura anatômica em função da deficiência hídrica são as seguintes: colapso das células, atraso na diferenciação dos tecidos, desorganização do feixe vascular e da epiderme e alterações na espessura do mesofilo e epiderme.

Palavras-chave:Eucalypus camaldulensis, deficiência hídrica, anatomia, cultura de tecidos

INTRODUCTION

Eucalyptus camaldulensis occurs along permanent and intermittent water streams in an extensive climatic zone of Australia (Gibson & Bachelard, 1994). The tree height varies according to the aridness of the region. Trees from Dimulah (Walsh River), Petford (Emu Creek) and Laura can reach 20 meters height. On the other hand, trees from the region between Mount Garmet and Georgetown are shorter, with heights that usually do not reach 10 meters (Barros & Novais, 1990).

Different authors have described the morpho-physiological variations existing between plants of E. camaldulensis from locations under different water deficit conditions (Gibson et al., 1994; Gibson and Bachelard, 1994; Alvear & Gutiérrez, 1995; Gibson et al., 1995; Farrell et al., 1996). However, few of them aimed to correlate the anatomy of this species to the different provenances in relation to the climatic and hydric conditions. (Pereira & Kozlowski, 1976; James & Bell, 1995).

The aim of this work was to obtain data showing the water deficit effects on the anatomical structure of tissues of E. camaldulensis shoots from different Australian provenances cultivated in vitro.

MATERIAL AND METHODS

Three different provenances of E. camaldulensis from distinct climatic locations were selected: Gilbert River (semi-arid latitude of 17°10' and longitude of 141°45' ), St. Katherin (dry tropical lat. of 14°30' and lon. of 132°15') and Petford (humid tropical lat. of 17°20' and long. of 144°58').

Explants consisting of an agglomerate of about 20 shoots each from previously established in vitro cultures were inoculated on basic MS medium (Murashige & Skoog, 1962) with different osmotic potentials (-0.5 as a control; -0.75, -1.5 and -3.0 MPa) obtained by the addition of various sorbitol concentrations (100 mM, 400 mM and 1000 mM) (Souza, 1997). After 20 days of culture under these conditions, stems and leaf blades from the median stem portion were collected for anatomical evaluation through the historesin inclusion tecnique (Kit Jung-Historesin) and light microscopy.

Sections of 4 mm were removed from the median portion of the stem and from leaf blades of each treatment and fixed in Mcdowell's solution during 48 hours under vacuum. The material was dehydrated in ethanol 70%, 96% and 100% for 2 hours in each step. Parainfiltration was done for 2 hours in a 1:1 100% ethanol solution plus liquid resin at room temperature and under vacuum, followed by 24 hours in a liquid resin solution (50 mL) plus 0,5g of activator powder. The samples were embedded in historesis in a polyethylene mold. The embedding mold was kept in a stove at 40°C until the complete hardening of the blocks.

The material was sectioned transversally in layers of 7 mm thickness using a microtome with a steel knife and stained with Toluidine Blue. Permanent slides were made in Entellan and photomicrographed using a Nikon camera (Labophot model).

RESULTS

The results of the anatomical evaluation of the transversal sections showed clear differences among treatments and controls (- 0.5 MPa) in all three provenances.

The Petford provenance

In the control, osmotic potential of -0.5 MPa, the leaf blade transversal sections (Figure 1.a) showed a differentiade adaxial and abaxial epidermis with well developed stomata. The chlorenchyma was clearly heterogeneous with distinct palisade mesophyll (PM) and spongy mesophyll (SM). The vascular bundle had distinct xylem and phloem (Figure 1.a). Under -0.75 MPa, SM and PM showed a smaller diffrentiation, with the SM being less distinct. There was a trend of formation of intercellular spaces in the SM. The adaxial epidermis presented turgid and rounded cells. In the vascular bundle, the cells were smaller and perfectly round with few stomata and a compact vascular bundle. (Figure 1.b). In the -1.5 MPa treatment, it was evident the formation of an aerenchyma and a lack of distinction between SM and PM with a homogenous chlorenchyma. The cells were turgid and the both epidermis were similar to the epidermis found on the -0.75 MPa treatment, although indistinctive in some points. The vascular bundle was less distinct and less organized (Figure 1.c). Under -3.0 MPa, the intercellular spaces nearlly disappeared, and the epidermis cells were more turgid and flattened with distinct stomata. The chlorenchyma was more heterogeneous with slight distinction between the PM and SM. The vascular bundle presented a larger wall thickening and a smaller diameter, compared to the other treatments. (Figure 1.d). TABLE 1 shows the quantitative analysis data about the plant leaf blade under water deficit.

Figure 1
- Micrographs of transversal sextions of leaf blade and stem of E. camaldulensis shoots from Petford provenance under in vitro induced water deficit (MPa). (PM=palisade mesophyll, SM=spongy mesophyll, VB=vascular bundle, C=chlorenchyma, M=medulla, Co=cortex, VC=vascular cilinder).

In the transversal sections of the stem (Figure 1.e-h), the -0.5 MPa control presented an epidermis with turgid and rounded cells and a cortex with large rounded cells. Some irregular shaped cells could be found. Few intercellular spaces were observed and the cells next to the vascular cambium were considerably smaller. The vascular bundle was normal with distinct xylem and phloem and an intact medulla (Figure 1.e). Large modifications occurred under the -0.75 MPa osmotic potential, where a total disintegration of the medulla was observed, as well as a complete disorganization of the other tissues. The cortex cells with very thin walls collapsed and the epidermis became irregular. The vascular cylinder was atypical and discontinuous, pushing the cortex against the epidermis (Figure 1.f). Under the -1.5 MPa potential, the tissues could be found in a higher level of organization, but the medulla still had a trend for disintegration. The cortex cells were rounded with little intercellular spaces, and the vascular cylinder was again continuous and better organized. However, the epidermis seemed to be irregular and almost indistinguishable from the cortex (Figure 1.g). In the -3.0 MPa osmotic potential, the epidermis was also indistinguishable from the cortex, which presented irregular shaped cells, but no collapsation. The vascular cylinder showed vessels of smaller diameter and more thickened walls. The medulla was normal (Figure 1.h).

The St. Katherine provenance

Of the three locations analysed, only shoots from S. Katherine presented a constant increase of sensitivity to the osmotic potential reduction. In the leaf, the control showed a normal aspect, similar to that found for Petford provenance shoots (Figure 2.a). Despite of the distinction among PM, SM and epidermis found on the -0.75 MPa osmotic potential treatment, some signs of sensitivity water deficit could be found, like the formation of evident intercellular spaces and atypical stomata. The adaxial epidermis showed turgid cells larger than those found on the abaxial epidermis. The SM presented dense cells, sometimes with more than one layer of cells (Figure 2b). Under the -1.5MPa treatment, the PM and SM were less differenciated with the presence of some large intercellular spaces. The vascular bundle presented irregular shaped cells with more thickened walls. The adaxial and abaxial epidermis presented flattened cells and rare and atypical stomata. The abaxial epidermis cells were smaller and rounded at the vascular bundle region (Figure 2.c). Under -3.0 MPa, there was a total disorganization of the leaf tissues. There was no distinction between PM, SM and epidermis, the bundle vessels were collapsed and the stomata were completely atypical. The chlorenchyma cells presented irregular shapes and sizes (Figure 2.d). The quantitative data analysis of the leaf blades is shown in the TABLE 2.

Figure 2
- Micrographs of transversal sections of leaf blade and stem of E. camaldulensis shoots from St. Katherine provenance under in vitro induced water deficit (MPa). (PM=palisade mesophyll), SM=spongy mesophyll, VB=vascular bundle, C=chlorenchyma, M=medulla, Co=cortex, VC=vascular cilinder).

In relation to the transversal section of the stem, the control presented a distinct epidermis with small cells, and a normal cortex with smaller cells next to the vascular bundle. The vascular cylinder was well organized with distinct liberian and xylem elements. The medulla was intact with large cells, reducing as closer to the vascular cylinder (Figure 2.e). Under - 0.75 MPa, the anatomical structure remained intact although a more irregular external outline of the stem was observed (Figure 2.f). A greater sensitivity to the water deficit was found in the 1.5 MPa. In this case, the stem shape was completely irregular, the medulla presented large ruptures and the vascular cylinder was discontinuous with compacted vessels. The cortex cells showed collapse signs with totally irregular shapes. The epidermis presented turgid cells, but not with a regular shape (Figure 2.g). In the osmotic potential of -3.0 MPa, the stem form was rounded and the medulla remained intact. The vascular cylinder was continuous with thickned wall vessels. The cortex cells were slightly irregular and the epidermis had small and round cells (Figure 2.h).

The Gilbert River provenance

The Gilbert River provenance presented the fastest responses to water deficit. In the leaf blade transversal sections, even under - 0.75MPa, the anatomical structure was completely altered in relation to the control shoots that presented a normal aspect, similar to other controls analysed before. (Figure 3.a and 3.b). There was no distinction between the PM and SM, the cells were totally irregular showing evident signs of collapse and there were no intercellular spaces. The adaxial and abaxial epidermis were completely irregular with flattened and disform cells showing clear signs of disintegration. The vascular region was collapsed without signs of reinforcement in the vessel walls (Figure 3.b). However, the leaf tissue under -1.5 MPa presented evident signs of cell turgescence. There was no differentiation between the chlorenchyma and the epidermis, and neither between PM and SM. The cells from the epidermic region showed clear signs of lignin and cellulose reinforcement, evidenciated by the green staining observed with Toluidine Blue (Johansen, 1940). The presence of intercellular spaces in the chlorenchyma was observed, as well as a disorganized and undifferentiated vascular bundle (Figure 3.c). In the - 3.0 MPa osmotic potential, the leaf tissues generally presented similar characteristics to those found on the shoots of St. Katherine provenance in the - 3.0 MPa. The epidermic cells were round shaped with thin walls, showing signs of disintegration. The chlorenchyma was homogeneous almost without intercellular spaces and presenting irregular shaped cells with more deformities closer to the vascular bundle, which was tightly compacted with indistinguishable xylem and phloem (Figure 3.d). In TABLE 3 the quantitative data analysis of the leaf blades under diffrent osmotic potencial conditions is presented.

Figure 3
- Micrographs of transversal sections of leaf blade and stem of E. camaldulensis shoots from Gilbert River provenance under in vitro induced water deficit (MPa). (PM=palisade mesophyll), SM=spongy mesophyll, VB=vascular bundle, C=chlorenchyma, M=medulla, Co=cortex, VC=vascular cilinder).

In the stem, the control presented a normal aspect (Figure 3.e). In the - 0.75MPa osmotic potential, the anatomical structure was greatly affected. The vascular cylinder was discontinuous, and its vessels had visible collapse signs. The cortex cells were withered with irregular shapes and showed a trend to form intercellular spaces. Although yet distinguishable, the epidermis was disorganized and with signs of lignin and cellulose reinforcement evidenciated by the stain (Johansen, 1940) (Figure 3.f). In the -1.5 and -3.0 MPa osmotic potential, the vascular cylinder were continuous. The cortex and the medulla in the -3.0 MPa treatment presented signs of a greater sensitivity to water deficit than those found in the -1.5 MPa treatment and showing irregular shaped cells and more intercellular spaces. In both cases, it was hard to precisely distinguish the epidermis cells from the cortex cells (Figures 3.g and 3.h).

DISCUSSION

Comparing the shoots from the three different provenances, the shoots from Gilbert River provenance presented the fastest and most evident responses to water deficit and more efficient adaptive anatomical mechanisms. This is probably due to its natural adaptation to a region with long periods of drought. These anatomical data corroborate the physiological and morphological behaviour pattern described in the literature (Gibson et al., 1994; Gibson & Bachelard, 1994; Alvear & Gutiérrez, 1995; Gibson et al., 1995, Farrel et al, 1996 ), although some irregularities could be found.

It can be clearly seen that the shoots of Petford provenance presented distinct behaviour to the water deficit reactions between leaves and stem. The leaf presented a more regular pattern. This could be seen as differences of stress sensibility between the leaf and the stem; as an indication of a metabolic behaviour deviation or as a strategic form of resistance to the water deficit (Levitt, 1972).

The quantitative data (TABLES 1, 2 and 3) indicated a trend to of clorenchyma related to osmotic potencial reduction. In material from Peford provenance, this increase was uniform up to -1.5 MPa, decreasing drastically under more acute water deficit. Although, this demean was also observed in material from Gilbert River provenance, under -3.0 MPa osmotic potencial, the mesophyll was thicker than the control (-0.5 MPa), suggesting a great resistance to water deficit. The material from St. Katherine provenance showed a uniform decrease of the chlorenchyma thickness from -0.75 MPa.

In relation to the epidermis, there was a trend of the adaxial epidermis to be thicker in all provenances in all treatments. However, there was not a pattern of response among the provenances in the epidermis thickness variation in relation to the variation of the osmotic potential. The increase of epidermis thickness is an indication of resistance to water deficit (Levitt, 1972).

According to James & Bell (1995), plants from the most arid regions present a thinner spongy mesophyll, a more thickened palisade mesophyll, larger chloroplasts and a lower cell density in the epidermis than plants coming from less arid regions. However, no striking general differences were found in the leaf morphology and anatomy of E. camaldulensis plants under different climatic condition. These characteristics were not so clear in the material analyzed in this current work, although the differences between the palisade and spongy mesophyll were observed in the control treatments (Figures 1a, 2a, 3a).

In other species, the water deficit also caused a reduction of the mesophyll thickness, like in grape (Dami & Hughes, 1995) and spring wheat (Zagdanska & Kozdoj, 1994). In Pinus sylvestris and Picea abies, the effects of the drought stress included the plasmolysis of mesophyll (Palomaki et al., 1995). However, in beech trees (Bussotti et al., 1995), potato, and tomato (Sam et al., 1996) it was observed an increase in mesophyll thickness in response to drought.

The responses, which were observed in shoots from all three provenances, ocurr faster and more efficienttly in plants belonging to regions where the water deficit is constant. However, it should be stated that the anatomical variations observed do not correspond to the adaptation pattern found on xeromorphic plants described by Esau (1977). This suggest that in spite of being from a semi-arid region, some trees of Gilbert River didn't show morphologically fixed adaptations.

In this work, the influence of the original environment of E. camaldulensis on its responses to the osmotic conditions at the anatomical level was evidenced.

CONCLUSIONS

Basically, the most typical anatomical alterations observed in response to the water deficit in the leaf blade were:

• epidermic cells tending to disintegrate in lower osmotic potencials (- 3.0 MPa);

• less differentiation between the palisade and spongy mesophyll;

• formation of intercellular spaces in the chlorenchyma observed in the intermediate osmotic potentials that tend to disappear in the extreme osmotic potentials;

• vascular bundle disorganization with greater compactation and thickness of the vessel walls and less distinction between the xylem and phloem.

In relation to the stem, the type of responses observed to the water deficit were the following:

• epidermis disorganization, becoming undistinguishable from the cortex;

• the cortex cells became irregular in shape and size;

• formation of intercellular spaces, although this was less frequent than the observed in leaves;

• vascular cylinder disorganization, presenting greater compactation and thickness of the vessel walls, as well as its existence in a discontinuous form in some cases;

• medulla disintegration.

ACKNOWLEDGEMENT

This research was supported by FAPESP-Fundação de Amparo à Pesquisa do Estado de São Paulo.

REFERENCES

ALVEAR, C.S.; GUTIÉRREZ, B.C. Crescimiento hasta los 42-44 meses de edad y estimación de parámetros genéticos de 23 procedencias e 196 famílias de Eucalyptus camaldulensis Denh. em cuatro sitios de la zona central de Chile. Ciência e Investigación Florestal, v.9, n.1, p.23-46, 1995.

BARROS N.F.; NOVAIS, R.F. Relação solo-eucalipto. Viçosa: Universidade Federal de Viçosa, 1990. 330p.

BUSSOTTI, F.; BOTTACCI, A.; BARTOLESI, A. et al. Morpho-anatomical alterations in leaves collected from beech trees (Fagus sylvatica L.) in conditions of natural water stress. Environmental and Experimental Botany, v.35, n.2, p.201-213, 1995.

DAMI, I.; HUGHES, H. Leaf anatomy and water loss of in vitro PEG-treated `Valiant' grape. Plant Cell, Tissue and Organ Culture, v. 42, n.2, p.179-184, 1995.

ESAU, K. Anatomy of seed plants. 2.ed. New York: John Wiley & Sons, 1977.

FARRELL, R.C.C.; BELL, D.T.; AKILAN, K. et al. Morphological and physiological comparisons of clonal lines of Eucalyptus camaldulensis: I. Responses to drought and waterloggin. Australian Journal of Plant Physiology, v.23, p.497-507, 1996.

GIBSON, A.; BACHELARD, E.P.; HUBICK, K.T. Growth strategies of Eucaliptus camaldulensis Dehn. at three sites in Northern Australia. Australian Journal of Plant Physiology, v.21, p.653-662, 1994.

GIBSON, A.; BACHELARD, E.P. Relationships between site characteristic and survival strategies of Eucalyptus camaldulensis seedlings. Australian Tree Species Research in China, p.91-95, 1994.

GIBSON, A.; BACHELARD, E.P.; HUBICK, K.T. Relationship between climate and provenance variation in Eucaliptus camaldulensis Dehn. Australian Journal of Plant Physiology, v.22, p.453-460, 1995.

JOHANSEN, D.A. Plant microtechnique. New York: McGrawhill Book, 1940. 532p.

JAMES, S.A.; BELL, D.T. Morphology and anatomy of leaves of Eucalyptus camaldulensis clones: variation between geographically separated locations. Australian Journal of Botany, v.43, p.415-433, 1995.

LEVITT, J. Responses of plants to enviromental stresses. New York: Academic Press, 1972 .

MURASHIGE, T.; SKOOG, F. A revised medium for rapid growth and bioassays tabacco tissue cultures. Physiologia Plantarum, v.15, p.473-497, 1962.

PALOMAKI, V.; HOLAPAINEN, J. K.; HOLAPAINEN, T. Effects of drought and waterlogging on ultrastructure of Scots pine and Norway spruce needles. Trees: structure and function, v.9, p.98-105, 1995.

PEREIRA, J.S.; KOZLOWISKI, T.T. Leaf anatomy and water relations of Eucalyptus camaldulensis and E. globulos seedlings. Canadian Journal of Botany, v.54, p.2868-2880, 1976.

SAM, O.; JEREZ, E.; VARELA, M. Caracteristicas anatomicas de hojas de apa (Solanum tuberosum L.) y tomate (Lycopersycon esculentum Mill.) can diferentes grados de tolerancia a estres de humedad y temperatura. Cultivos Tropicales, v. 17, n.2, p.32-38, 1996.

SOUZA, G.M. Influência da deficiência hídrica no desenvolvimento morfogenético do Eucalyptus camaldulensis Dehn. in vitro. Piracicaba, 1997. 70p. Dissertação (Mestrado) - Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo

ZAGDANSKA, B.; KOZDOJ, J. Water stress-induced changes in morphology and anatomy of flag leaf of spring wheat. Acta Societatis Botanicorum Poloniae, v.63, n.1, p.61-66, 1994.

Received May 14, 1998

Accepted April 20, 1999

• ALVEAR, C.S.; GUTIÉRREZ, B.C. Crescimiento hasta los 42-44 meses de edad y estimación de parámetros genéticos de 23 procedencias e 196 famílias de Eucalyptus camaldulensis Denh. em cuatro sitios de la zona central de Chile. Cięncia e Investigación Florestal, v.9, n.1, p.23-46, 1995.
• BARROS N.F.; NOVAIS, R.F. Relaçăo solo-eucalipto Viçosa: Universidade Federal de Viçosa, 1990. 330p.
• BUSSOTTI, F.; BOTTACCI, A.; BARTOLESI, A. et al. Morpho-anatomical alterations in leaves collected from beech trees (Fagus sylvatica L.) in conditions of natural water stress. Environmental and Experimental Botany, v.35, n.2, p.201-213, 1995.
• DAMI, I.; HUGHES, H. Leaf anatomy and water loss of in vitro PEG-treated `Valiant' grape. Plant Cell, Tissue and Organ Culture, v. 42, n.2, p.179-184, 1995.
• FARRELL, R.C.C.; BELL, D.T.; AKILAN, K. et al. Morphological and physiological comparisons of clonal lines of Eucalyptus camaldulensis: I. Responses to drought and waterloggin. Australian Journal of Plant Physiology, v.23, p.497-507, 1996.
• GIBSON, A.; BACHELARD, E.P.; HUBICK, K.T. Growth strategies of Eucaliptus camaldulensis Dehn. at three sites in Northern Australia. Australian Journal of Plant Physiology, v.21, p.653-662, 1994.
• GIBSON, A.; BACHELARD, E.P. Relationships between site characteristic and survival strategies of Eucalyptus camaldulensis seedlings. Australian Tree Species Research in China, p.91-95, 1994.
• GIBSON, A.; BACHELARD, E.P.; HUBICK, K.T. Relationship between climate and provenance variation in Eucaliptus camaldulensis Dehn. Australian Journal of Plant Physiology, v.22, p.453-460, 1995.
• JOHANSEN, D.A. Plant microtechnique New York: McGrawhill Book, 1940. 532p.
• JAMES, S.A.; BELL, D.T. Morphology and anatomy of leaves of Eucalyptus camaldulensis clones: variation between geographically separated locations. Australian Journal of Botany, v.43, p.415-433, 1995.
• LEVITT, J. Responses of plants to enviromental stresses New York: Academic Press, 1972 .
• MURASHIGE, T.; SKOOG, F. A revised medium for rapid growth and bioassays tabacco tissue cultures. Physiologia Plantarum, v.15, p.473-497, 1962.
• PALOMAKI, V.; HOLAPAINEN, J. K.; HOLAPAINEN, T. Effects of drought and waterlogging on ultrastructure of Scots pine and Norway spruce needles. Trees: structure and function, v.9, p.98-105, 1995.
• PEREIRA, J.S.; KOZLOWISKI, T.T. Leaf anatomy and water relations of Eucalyptus camaldulensis and E. globulos seedlings. Canadian Journal of Botany, v.54, p.2868-2880, 1976.
• SAM, O.; JEREZ, E.; VARELA, M. Caracteristicas anatomicas de hojas de apa (Solanum tuberosum L.) y tomate (Lycopersycon esculentum Mill.) can diferentes grados de tolerancia a estres de humedad y temperatura. Cultivos Tropicales, v. 17, n.2, p.32-38, 1996.
• SOUZA, G.M. Influęncia da deficięncia hídrica no desenvolvimento morfogenético do Eucalyptus camaldulensis Dehn. in vitro Piracicaba, 1997. 70p. Dissertaçăo (Mestrado) - Escola Superior de Agricultura "Luiz de Queiroz", Universidade de Săo Paulo
• ZAGDANSKA, B.; KOZDOJ, J. Water stress-induced changes in morphology and anatomy of flag leaf of spring wheat. Acta Societatis Botanicorum Poloniae, v.63, n.1, p.61-66, 1994.

Publication Dates

• Publication in this collection
  17 Sept 1999
• Date of issue
  July 1999

History

• Received
  14 May 1998
• Accepted
  20 Apr 1999