Problems Encountered in the Culture of Isolated Pollen of a Burley Cultivar of Nicotiana tabacum

Pollen isolated from anthers of a Burley cultivar of N. tabacumby an anther homogenization procedure failed in culture to retainviability and produce plantlets even when isolation was precededby 14 d of anther preculture. By surgical manipulation of anthersprior to their culture it was shown that pollen viability wasvery sensitive to injury caused to the anther wall. Plantletscould be obtained however by culture of anthers in which oneor both pollen sacs had been carefully opened along the dehisenceline (DL). The somewhat lower yield of plantlets from such antherscompared to that from intact anthers is considered to resultnot from any change in the atmospheric environment of the pollenas a result of opening the pollen sac but from the inabilityin every case to keep the incisions strictly along the dehiscenceline. Pollen isolated from 14 d precultured anthers by this DL techniquewas able to yield plantlets when cultured in a simple definedmedium. However DL-isolated grains from 4 d precultured anthersfailed to retain viability in culture. It is concluded that pollen of this cultivar suffers severeinjury when isolated by the homogenization method and that thiscan be overcome by the DL technique. However conditions of culturehave not been established which reproduce for this cultivarthe nutritional conditions operating within the anther duringits early period in culture and which are essential to plantletformation.     

Shed Pollen Culture in Hordeum vulgare

Anthers of Hordeum vulgare cv. Sabarlis at the mid-unicellularpollen stage, pretreated in the excised spike for 14 d at 7°C, dehisce within 24 h of being floated on the surfaceof liquid medium. About half the pollen (1500 grains per anther)is liberated into the medium. If the anthers are then removedand the cultures re-incubated, calluses develop from the shedpollen in high yields. At low anther densities, 10p–20(1–3 x 10⁴ grains) per ml, medium preconditioned by anthersand supplemented with m-inositol (1000 mg 1^–1) is required,but at high densities, 120 anthers (2 x 10⁵ grains) per ml,preconditioning is less important, the cultured anthers themselveshaving a sufficient conditioning influence. Large-scale dissectionof anthers can be avoided by use of drops of medium, the volumebeing increased gradually as culture proceeds. Pollen remainingin the anthers after 3 d gives rise to calluses if isolatedmechanically and cultured in the inositol medium. The use ofshed pollen is seen as particularly valuable for culture inspecies whose anthers are small, tedious to dissect out anddifficult to process without severe damage.     

Culture of Barley Anthers in Conditioned Media

High yielding anther cultures of Hordeum vulgare cv. Sabarlisare obtained at inoculation densities of 10—20 per mlby use of media previously conditioned by Sabarlis anthers.To achieve these high yields anthers at the mid-unicellularpollen stage (stage 2), stressed in the excised spike for 14d at 7 °C, are necessary, but for conditioning of mediaolder anthers may be used with or without the stress pretreatment.Conditioning for 7 d by anthers at young bicellular pollen stages(stages 5—6) is highly effective. Hormones supplied inthe medium interact synergistically with the conditioning factor. Sabarlis ovaries are shown to be even more effective for conditioningthan anthers, whereas glumes and other parts of the spike arerelatively ineffective. Anthers of oats, rye, wheat, maize,tobacco, and one other genotype of barley are also less effectivethan Sabarlis anthers. The improved method of anther culture is more efficient thanspike culture for the production of pollen callus in Sabarlisbarley.     

Effects of Physical and Chemical Factors on Induction Frequency of the Pollen Plantlet and Changes in Starch Formation in Anthers

Physical and chemical facttors affected distinctly induction frequency of the pollen plantlets from anther culture in vitro of Lycium. Experimental results showed that the anthers with their pollen grains at the uninucleate stage when the nuclei were centrally situated, were the best material for the anther culture. The different proportions of various hormones have affected the embryoid formation. When the MS medium was supplemented with 6-BAP (1 ppm) and NAA (0.1 ppm), the induction frequency was increased ( 16.9% ). When 3–15 per cent of sucrose was added in medium, the embryoids were induced and 15 per cent was the optimum. The callus of the filament was inhibited by the increase of the sucrose concentration. Before inoculation the anthers were pretreated at 3–5℃ for 4 days, the frequency of embryoid formation was efficiently increased in comparision with those of untreated anthers. The induction frequency of normal anthers was only 2.8 per cent, but that of the anthers pretreated was 3–9 per cent, the highest was 8.9 per cent .The changes of substances in anther pretreated and in normal anther was compared by means of histochemistry. Under normal conditions, there were a lot of starch accumulated in the inner wall Of the anthers and the distribution of the cytoplasm and the staining of the protein were even: In the anther pretreated, the starch grains have disappeared and the cytoplasm has condensed and the staining of the protein wasn't even. The differences may be related to induction, frequency of the anther culture.     

Temperature-stress Pretreatment in Barley Anther Culture

Methods of pretreating anthers at different temperatures priorto culture have been tested, with respect to pollen-callus productionand plant regeneration, in Hordeum vulgare cv. Sabarlis. For callus production, pretreatment of excised spikes (in sealedPetri dishes) was more effective than pretreatment of excisedtillers (in water or in polythene) at both 4 and 25 °C.Pretreatment of individual anthers at these temperatures wasdeleterious. Greater callus yields resulted from pretreatmentat 4 than at 25 °C, both for spikes and tillers, 3–5weeks being required for maximal yields at 4 °C and 3–5days at 25 °C. At 4 °C, a shorter pretreatment was requiredfor spikes than for tillers. Pretreatment of spikes was alsomore effective at 4 than at 7, 14 or 20 °C. Pretreatmentof individual spikelets at 4 °C was as effective as thatof whole spikes. For plant regeneration, calluses derived from pretreatment ofspikes were more effective than those derived from pretreatmentof tillers. More plants resulted from pretreatment at 4 thanat 25 °C, both for spikes and tillers. Maximal pretreatmenttimes for plant regeneration generally exceeded those for callusproduction. Following spike pretreatment at 4 °C the maximumfor plant regeneration exceeded that for callus production byabout 2 weeks. With this optimal pretreatment approximately60 per cent of the calluses gave rise to plantlets. Among this60 per cent, for every three calluses giving albinos, two gavegreen plantlets, equivalent to five green plantlets on averagefor every 100 anthers (= two spikes) cultured. The ratio ofgreen to albino plantlets was lower for all other pretreatments. Hordeum vulgare L., barley, anther culture, pollen callus, pollen plant-production, temperature stress     

The anther-culture response of triticale line x tester progenies

Seven triticale cultivars (Ampiac, Aubrac, Trinidad, Ticino, Lamberto, Pronto and Prado) and their F1 hybrids obtained after crossing in a line x tester scheme were examined with respect to their androgenetic effectiveness. The embryo induction rate (number of embryos per 100 anthers), green plant regeneration rate (number of green plantlets per 100 embryos), plant yield (number of green and albino plantlets per 100 anthers) and green plant yield (number of green plantlets per 100 anthers) were assessed. The multivariate and univariate effects of general (GCA) and specific (SCA) combining abilities for the studied traits were estimated and tested. Significant differences between the genotypes were found for individual traits as well as for all the traits treated jointly. Hybrids generally showed a better response in anther culture than their parental genotypes. Heterosis effects were observed in some hybrids for embryo induction rate and green plant yield. GCA and SCA variances were significant and a dominance of the GCA over the SCA variation was found. Among the examined cultivars, Ticino and Pronto were characterised by positive and significant GCA for embryo induction and green plant yield, and these cultivars may be recommended for the improvement of anther culture responsiveness in triticale.     

Cold-pretreatment of Excised Flower Buds in Float Culture of Tobacco Anthers

Pretreatment of excised flower buds at 7–9 °C is shownto be more effective in float culture of Nicotiana tabacum anthersthan pretreatment at 5 °C. The small temperature differenceresults in greatly enhanced embryo yields when the treatmentis given just before (stage 3), during (4) or just after (5)the first pollen division. Anther productivity (total embryoyield per anther) increases with the time of pretreatment toa peak beyond which further pretreatment is deleterious. Thepeak is attained sooner at 9 than at 7 °C and sooner inanthers pretreated at stage 5 than at stage 3. Peak productivityappears always to be the same. Owing to batch variations, accuratepretreatment-times cannot be specified. Suggested mean timesfor buds of mixed stage are about 12–14 days at 7 °Cand about 7–9 days at 9 °C. Anthers dehisce in culture, the time to dehiscence decreasingwith increasing anther stage. Mid-stage-5 anthers open withinthe first few days, sooner with pretreatment at 9 than at 7°C, and high-yielding free pollen cultures can be obtainedwithout mechanical disruption of the anthers. Buds survive longest when harvested at the beginning of theearly flowering period and kept at 7 °C. It is proposedthat the pretreatment exerts its effect not by altering thecourse of the first pollen division, as suggested by others,but by delaying anther deterioration and thus assuring survivalof a greater proportion of the pollen grains switched into embryogenesis. Nicotiana tabacum L., tobacco, anther culture, embryogenesis     

Differential development of plastids during microspore embryogenesis in barley

Summary In order to understand and limit albino plantlet formation during pollen embryogenesis in barley (Hordeum vulgare L. cv. Igri), plastid feature was followed during pollen embryogenesis under two anther culture conditions and compared to plastid development in the zygotic embryo. The first condition was characterized by cold pretreatment and maltose in the induction medium. Both embryos and calli were then obtained. During pollen embryo development, up to 30% of plastids had abnormal features. Disruptions mainly affected the plastid size, the feature of plastid envelopes, thylakoid and granum organization, as well as starch accumulation. In pollen calli, superficial cells had meristematic features. Up to 50% of plastids exhibited the above mentioned abnormalities. Internal cells were highly vacuolated with amyloplast-like plastids; envelopes had normal features but no internal membrane was detected. Pollen embryo-derived plantlets had a green-to-albino ratio (G/A) being equal to 1.0, whereas calli-derived embryos only formed albino plantlets. The second condition was characterized by mannitol pretreatment and the presence of both maltose and mannitol in the induction medium. No callus was formed but most of microspore-derived structures developed haploid embryos and then the green plantlets (200 plantlets per 100 responding anthers, G/A=9.4). In this case, plastid development in zygotic and pollen embryos were similar and almost no albino plantlets were formed.     

Influence of Preculture Variables on Microspore Embryo Production in Brassica napus ssp. oleifera cv. Duplo

The effect of four preculture variables on microspore embryoinduction and growth were examined: (1) the source of the budselected for culture (apical or axillary inflorescence; (2)the method of harvest (single harvest of whole inflorescenceor sequential harvest of individual buds; (3) the length ofthe bud (2, 3 or 4 mm); and (4) the application of a 4 ^°Cpretreatment to the bud after harvest. Microscopic and macroscopicanalysis of every anther used for culture permitted an assessmentof the following parameters: (1) the percentage of induced buds;(2) the number of induced anthers per induced bud; (3) the numberof productive buds (with macroscopic embryos) as a percentageof the induced buds; (4) the degree of induction per inducedanther (an estimate of the number of microspores in which initialembryogenic divisions had commenced); and (5) embryoid survival(the number of embryos as a proportion of the degree of induction). The product of parameters 1 and 2 gave the number of inducedanthers and all five parameters were components of the finalyield - the number of embryos produced per bud cultured. It was found that the maximum number of induced buds (67·0per cent) occurred with 2 mm sequentially harvested non-pretreatedbuds. Overall, the values decreased with increasing bud lengthand were lower for pretreated and axillary buds. In contrast,the two other estimates of induction - number of induced anthersper induced bud and degree of induction per induced anther -both had maximum values from 3 mm sequentially harvested, pretreatedbuds from apical inflorescences. The highest final yield ofembryos per cultured bud (44·9) was found with 2 mm non-pretreatedbuds taken from a single harvest of the apical inflorescence.The study therefore confirmed that the different componentsof the final embryo yield are differentially affected by thefour preculture variables tested. These variables must be controlledif reproducible results are to be achieved. Brassica napus, tape, anther culture, pollen, microspore, haploid     

Embryoid Formation in Pollen Grains of Nicotiana tabacum

Anthers of Nicotiana tabacum (n = 24) were cultured on nutrientagar and examined at intervals for pollen embryoids. Embryoidswere formed in anthers of varying developmental stage, the youngestof which coincided with the liberation of free microspores fromtetrads, and the oldest with the formation of bicellular grains.This period in the development of the anther occupied 4–5days. Older anthers within this range were more successful thanyounger anthers. The first mitosis of the pollen was typicallyasymmetric and resulted in the formation of unequal generativeand vegetative cells. Some of the grains then went into a lagphase for at least 5–6 days, after which the mitotic conditionwas restored. Embryoids were formed by repeated division ofthe vegetative cell. If the generative cell divided, it didso only once or twice. Occasionally the first mitosis was symmetricand gave rise to equal cells, and in these instances both cellsprobably participated in embryoid formation. The youngest anthersexamined were probably less successful because fewer grainssurvived to enter mitosis. The number of embryoids produced varied considerably from oneanther to another both within the same bud and between differentbuds: values ranging from less than 400 to 10 000 per antherwere encountered. Most of these degenerated after the firstfew divisions, partly because they burst prematurely from thepollen grain wall. Embryoids which continued to develop formedplantlets and/or callus. The largest number of plantlets obtainedfrom one anther was 32. Haploid plantlets were also regeneratedfrom callus by transferring it to a low-sugar medium withoutauxin. The behaviour of grains not forming embryoids was also noted.     

Role of Sucrose in Microspore Embryo Production in Brassica napus ssp. oleifera

Dun well, J. M. and Thurling, N. 1985. Role of sucrose in microsporeembryo production in Brassica napus ssp. oleifera.—J.exp. Bot. 36: 1478–1491. One cultivar of winter oil seed rape (Brassica napus ssp. oleifera)and three cultivars of spring rape were used in a study of theeffects of sucrose on microspore survival and embryo inductionin cultured anthers. A preliminary study on the winter cultivar(Fiona) revealed that the osmotic pressure of the supernatantof anther homogenates was equivalent to a solution of 17% sucrose.A study of microspore survival and embryo induction in thiscultivar on media containing either 8 %, 12%, 16% or 20% sucroserevealed the highest survival (after 16 d) and the greatestnumber of anthers with induced embryos (after 42 d) occurredon the highest sucrose concentration. A subsequent study on three spring cultivars (Willi, Duplo andTower) examined microspore survival at 8 d and embryo induction(42 d) on media containing either 8 % or 16 % sucrose and againrevealed much higher survival and induction at the higher concentration.The variation in response between the cultivars was also reducedby culture at the higher sucrose concentration. The beneficialeffect of the 16% level occurred regardless of the growth environmentof the donor plants and of the stage of pollen development atthe start of culture. However, macroscopic embryos emerged onlyfrom anthers on the 8 % sucrose concentration, suggesting thattransfer of anthers from a high to a normal sucrose concentrationduring culture would ensure that full advantage was taken ofthe much higher initial survival on the higher concentration Key words: Brassica napus, sucrose, microspore embryo production     

Pollen Dimorphism and Its Relation to the Formation of Pollen Embryos in Anther Culture of Wheat (Triticum aestivum)

The anomalous pollen grains in vivo at the frequencies of 2.61% and 6.81% respectively were found in anthers of two hybrid materials of wheat from which we had obtained the higher frequencies of induction of pollen embryos in anther culture in the past few years. This indicated that the pollen dimorphism also presented itself in the anthers on wheat plants. A comparative cytological study of development of anomalous pollen grains in vivo and of the early stages of the formation of pollen embryos in vitro was carried out and the results suggest that in cytological details there is an obvious similarity between them. This reveals the possibility that the pollen embryos night derive itself from the anomalous pollen grains in wheat as in the other plants. Therefore, it was considered that the appearance and frequency of the anomalous pollen grains could show the senibility of the development of microspores to the changes of various internal and external factors. The significance of the sensibility for the formation of pollen embryos in anther culture of wheat is discussed.     

Development of embryos and seedlings from pollen grains inLycium halimifolium Mill. in thein vitro culture

Anthers ofLycium halimifolium were grownin vitro at the following stages of development: tetrads, microspores and binucleate pollen grains. Pollen plantlets were obtained only from anthers inoculated at the stage of microspores. The growth of androgenic embryos was very slow. Mature embryos were formed in about 3% of the inoculated anthers. The largest number of embryos obtained from one anther was 4.     

Disposition of Pollen in situ and its Relevance to Anther/Pollen Culture

Disposition of pollen in immature anthers of Hordeum vulgareis illustrated by scanning and transmission electron microscopy.Freeze-fracture confirms that the pollen is confined to a uniseriatecolumn aligned against the tapetum. There is no free pollenin the lumen of the anther loculi. In contrast, in Nicotianatabacum and Paeonia lactlflora the pollen is disposed at randomand occupies the whole of each loculus. Freezing preserves thefluid content of the loculi, appearing in fracture profilesas an amorphous matrix in which the pollen is embedded. Thematrix, which generally obscures the tapetum, is present throughoutthe microspore phase but diminishes as the spores enlarge. Itis still present in N. tabacum and H. vulgare at the first pollendivision, fragmentary at this stage in P. lactiflora, but isno longer discernible in any of the species at the onset ofpollen-grain maturation. Pretreatment of excised Hordeum spikes at 4 ?C during the microsporephase, a prerequisite for anther/pollen culture, disrupts thenormal developmental sequence but does not alter the uniseriatedisposition. Before the spores start to divide, however, thetapetum degenerates and the fluid phase is dispersed. The observations are discussed in relation to isolated pollenculture, float culture of anthers and the switch in programmefrom gametophytic to sporophytic development. Key words: Pollen, Tapetum, Freeze-fracture     

Factors affecting the induction of pollen plants of intergeneric hybrids of Triticum aestivum X Triticum-Agropyron

Summary Experimental results showed that the use of potato extract as a basic component of culture medium had a promoting effect on producing calli in anther culture of the intergeneric hybrids of Triticum aestivum × Triticum-Agropyron (intermediate type). The induction frequencies of pollen callus on the Potato-II medium containing potato extract as the main component was much higher than that found on N₆ and W₅ media. The induction frequencies of pollen callus and green plantlets in four intergeneric hybrid material inoculated at the late-uninucleate pollen stage were all higher than those inoculated at the mid-uninucleate stage. Appropriate increases in culture temperature significantly increased pollen callus induction frequencies of the intergeneric hybrids. The genotype and physiological state of anther donor plants also influenced pollen callus and green plantlet induction frequencies.     

Anther Factor(s) in Barley Anther Culture

The effects of anther tissues were studied systematically on microspores forming multicellular units and furthermore on pollen callus formation in the anther culture of Hordeum vulgare (cv. Sabarlis). Anther productivity was found to be greatly enhanced by use of medium previously conditioned by anthers. In 15 experiments observed, anthers produced 26 times on average more calli in the conditioned medium than in control, in a few cases, even more than 80 times more calli formed. According to this, the authors supposed that cultured anthers released some components, anther factor (s) (AF), which is important to androgenesis in the culture. To achieve high yields of callus, culture was restricted to anthers which had been subpected to cold pretreatment. The temperature stress could not be replaced by the AF. However, for conditioning medium, anthers at binuclear stage were found to be more effective than the test anthers either with or without the pretreatment. Anthers from other 8 barley varieties were also effective for conditioning, as the difference of anther productivity still existed in the culture with conditioned medium between various genotypes tested. Anther response and callus yield were increased in both the culture of anthers at mid and late-uninuclear stage by use of conditioned medium. AF interacted synergistically with m-inositol. Cytological observation showed that AF increased apparently the formation of MPGs, while m-inositol mainly stimulated callus formation from MPGs. To some extent, the effect of exogenous hormone(s) could be replaced by AF. The anther response and pollen callus yield could be much enhanced by increasing anther inoculation density, which also raised the AF level in the culture. Thus, by use of the temperature stress prior to anther culture and culture of test anthers in conditioned medium with m-inositol, or at higher inoculation density, a very high production of pollen callus could be obtained in barley anther culture. For meeting the more specialized requirements of less responsive species or genopypes, the principles given here may be provide some basic information.     

Donor Plant Growth Factors Affecting Anther Culture of Maize and Sweetcorn (Zea mays)

Anthers from seven unselected commercial sweetcorn lines andten experimental maize lines were cultured on a liquid/solidbi-layer culture medium, containing 13 % maltose as the carbonsource. Mean anther efficiencies (number of embryos per 100anthers plated) of 0 to 27.6 % were recorded, with the maximumefficiency of 57.1 % from one plant. The anther efficiency wasfound to be dependent on genotype, microspore developmentalstage and the growth temperatures of donor plants. Immaturemicrospores were found to continue their development duringthe cold pretreatment of the spikes, and this in turn influencedthe level of response to culture. Direct regeneration of embryoidsto plants occurred most frequently when well formed uni- orbi-polar embryos were produced. The quality of embryo producedwas apparently inversely correlated with the number of embryosproduced. Zea mays, haploid culture, embryos, microspores     

Haploid plants from in vitro anther culture of the leguminous tree,Peltophorum pterocarpum (DC) K. Hayne (Copper pod)

The development of haploid callus, embryos and plantlets from cultured anthers and the various factors affecting androgenesis in Peltophorum pterocarpum (Copper pod), a tropical legume tree is reported. A pretreatment of flower buds at moderate temperature of 14°C for 8 days was most effective for callus production. The colour of the anther was found to be a reliable and efficient indicator for identification of suitable stage of anther for culture. The frequency of anthers which produced callus and shoots was highest when anthers were cultured at mid or late-uninucleate stage. A high sucrose concentration of 10% is a specific media requirement for androgenesis. The haploid nature of the embryos, callus and regenerated plants (n=14) were confirmed by chromosome count.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid - BAP bezylaminopurine     

Inheritance of anther culture derived green plantlet regeneration in wheat (Triticum aestivum L.)

A study was set up to determine the inheritance and combining ability of the factors anther culture response and green plant regeneration. Reciprocal crosses were made between cultivar Ringo Sztar, showing high anther culture response and the cultivars Ciano 067 and Benoist H77022, showing a high level of green plant regeneration. Averaged over all genotypes, 23.0% of the anthers responded and a callus induction frequency of 77.8% was observed. Of all the embryos, 43.0% developed into plantlets, 25.6% of the regenerants being green, the result being that 3.3 green plants per 100 anthers were formed. Genotypic effects accounted for 57.7%, 86.3% and 77.5% of the total variance of anther culture response, callus induction frequency and embryo induction frequency, respectively. Additive and dominant gene action was detected for all characteristics, including green plant regeneration. No reciprocal differences were found for anther culture response, embryo induction frequency and green plant regeneration, indicating no cytoplasmic effects. A small but significant reciprocal difference was found for callus induction frequency. Embryo production was primarily correlated with anther culture response and not with the number of embryos produced per plated anther or per responding anther. Possible mechanisms for the inheritance of green plant regeneration are discussed.Abbreviations CIRA callus induction frequency per responding anther - ERA embryo induction frequency per responding anther - FHB fusarium head blight - MS-medium Murashige & Skoog (1962) medium - REML residual maximum likelihood     

Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.)

In order to better understand androgenic albinism in barley, we compared plastid differentiation during anther culture in two cultivars, an albino (spring cultivar Cork) and a non-albino (winter cultivar Igri) producing cultivar. The ultrastructure of plastids and the relative amount of DNA containing plastids were followed in both cultivars during the androgenic process and correlated with the proportion of regenerated chlorophyllous plantlets. For androgenesis, anthers were collected at the uninucleate stage, during mid- or late-microspore vacuolation. At this stage DNA was detected in 15.3 ± 2. 7% of microspore plastid sections in the winter cultivar Igri, compared to 1.7 ± 0.5% in the spring cultivar Cork. In the winter cultivar Igri, starch was broken down after anther pretreatment but plastids divided rapidly during anther culture and thylakoids developed in the stroma. Prior to regeneration, plastids contained 2.0 ± 0.2 thylakoids per plastid and starch represented 26.1 ± 3.3% of the plastid volume. In the spring cultivar Cork, plastids followed a different developmental pathway. After anther pretreatment, microspore plastids differentiated exclusively into amyloplasts, accumulating starch and losing their thylakoids as well as their capacity to divide. This developmental pattern became progressively more marked, so that by the end of anther culture plastids contained 0.5 ± 0.4 thylakoids per plastid and starch represented up to 90.3 ± 4.3% of plastid volume. Following androgenesis, the response was similar in both cultivars except that the winter cultivar Igri provided 87.8% of chlorophyllous plantlets compared to 99.7% albino plantlets in the cultivar Cork. The results presented here suggest that the exclusive regeneration of albino plantlets in the spring cultivar Cork may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions. Received: 13 March 2000 / Revision accepted: 6 June 2000