Effect of bialaphos and phosphinothricin on plant regeneration from long- and short-term callus cultures of Gladiolus

Summary Callus was initiated from in vitro-grown plants of Gladiolus cultivars ‘Jenny Lee’ and ‘Florida Flame.’ The age of callus used for regeneration of plants was either 9 mo. old or 8 yr old from ‘Jenny Lee,’ and 4 mo. old from ‘Florida Flame.’ Regeneration medium consisted of Murashige and Skoog’s basal salts medium supplemented with 2.0 mg/l (9.3 μM) kinetin. This medium was supplemented with various concentrations of either bialaphos (Meiji Seika, Tokyo, Japan) or phosphinothricin (Hoechst-Roussell, Frankfurt, Germany). Bialaphos was more effective than phosphinothricin at stimulating plant regeneration. Plants regenerated from 8-yr-old callus of ‘Jenny Lee’ only when the regeneration medium was supplemented with 0.10 mg/l bialaphos. A bialaphos concentration of 0.01 mg/l stimulated regeneration from 9-mo.-old callus of cultivar ‘Jenny Lee’ and 4-mo.-old callus of ‘Florida Flame.’     

Callus induction and plant regeneration from broccoli (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. italica) for transformation

We evaluated the efficiency of callus induction and plantlet regeneration from hypocotyl explants of broccoli (Brassica oleracea var. italica). The cultivars were ‘Marathon’, ‘Greenbelt’, and ‘Shogun’. Transformation success was not affected by the presence of tobacco feeder-cell layers on the culture media. The frequency of shoot regeneration was greater from 10-d-old hypocotyls than from 14-d-old hypocotyls. Both ‘Marathon’ and ‘Greenbelt’ had higher potentials for tissue regeneration than did ‘Shogun’. We found that for transformation selection, the optimum concentration was either 50 mg/L kanamycin or 100 mg/L genetkin.     

Response of durum wheat cultivars to immature embryo culture,callus induction and in vitro salt stress

Response of twenty eight cultivars of durum wheat (Triticum turgidum var. durum) to immature embryo culture, callus production and in vitro salt tolerance was evaluated. For assessment of cultivars to salt tolerance, growing morphogenic calli were exposed to different concentrations of NaCl (0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 and 2.1% w/v) added to the culture medium during two subsequent subcultures (4 weeks each). Comparison of cultivars for callus induction from immature embryo was based on callus induction frequency and fresh weight growth of callus (FWG). While, for salt tolerance, the relative fresh weight growth (RFWG) and necrosis percent of callus were used. There were significant differences among cultivars for potential of regeneration from immature embryo, and ‘Shahivandi’ a native durum wheat cultivar originating from western Iran was superior among the cultivars tested. The FWG distinguished cultivars more than callus induction frequency did for callus induction evaluation. Hence, a range of FWG from 1.23 to 14.65 g was observed in ‘Mexical-75’ and ‘Omrabi-5’ cultivars, respectively. Growing calli derived from cultivars ‘PI 40100’ and ‘Dipper-6’ showed superiority for tolerating salinity under in vitro conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of genotype and medium composition on linseed (<Emphasis Type="Italic">Linum usitatissimum</Emphasis>) ovary culture

Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders to develop new cultivars in a shorter time period. Many research groups successfully created new linseed genotypes through anther culture; however ovary culture has been the subject of only a few earlier studies. In the present study, the effect of genotype and growth regulators combination on callus induction and shoots regeneration in ovary culture of nine commercially important linseed cultivars was investigated. Ovaries were cultured on modified MS medium supplemented with three different combinations of plant growth regulators. Variable callogenic responses were expressed by all of the genotypes tested on different induction media. The results suggested that specific combination of growth regulators for callus induction must be designed for each genotype. Shoot regeneration from ovary derived callus is a critical phase of the whole gynogenetic process. Differences in adventitious shoot formation frequency among genotypes were demonstrated and four responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’ manifested the highest adventitious shoot formation frequency with a high number of shoots per explant. The optimum ratio of growth regulators for shoot regeneration was shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the highest shoot regeneration frequency when callus had originated on induction medium supplemented with 2 mg L⁻¹ BAP and 2 mg L⁻¹ NAA, while combination of 1 mg L⁻¹ BAP and 2 mg L⁻¹ IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest rate of shoots per explant has been obtained in second subculture.     

Effects of carbon source and concentration on development of lingonberry (<Emphasis Type="Italic">Vaccinium vitis-idaea</Emphasis> L.) shoots cultivated <Emphasis Type="Italic">in vitro</Emphasis> from nodal explants

Summary Carbohydrate type and concentration and their interactive effects on in vitro shoot proliferation of three lingonberry (Vaccinium vitis-idaea ssp. vitis-idaea L.) cultivars (‘Regal’, ‘Splendor’, and ‘Erntedank’) and two V. vitis-idaea ssp. minus (Lodd) clones (‘NL1’ and ‘NL2’) were studied. Nodal explants were grown in vitro on medium with 2 μM zeatin and either glucose, sorbitol, or sucrose at a concentration of 0, 10, 20, or 30 gl⁻¹. The interactive effects of carbohydrate type and concentration and genotype were important for shoot proliferation. The best response was afforded by sucrose at 20 gl⁻¹ both in terms of explant response and shoot developing potential, although glucose supported shoot growth equally well, and in ‘NL1’ at 10 gl⁻¹ it resulted in better in vitro growth than sucrose. Carbohydrate concentration had little effect on shoot vigor. The genotypes differed in terms of shoots per explant, length, and vigor, leaves per shoot, and callus formation at the base of explants; this was manifested with various types and concentrations of carbohydrate. Changing the positioning of explants on the medium from vertically upright to horizontal increased the shoot and callus size, but decreased shoot height and leaves per shoot. Proliferated shoots were rooted on a peat:perlite (1∶1, v/v) medium and the plantlets were acclimatized and eventually established in the greenhouse.     

The establishment of cell suspension cultures ofGladiolus that regenerate plants

Summary Inflorencence stalks from greenhouse-grownGladiolus plants of the cultivars ‘Blue Isle’ and ‘Hunting Song’ cultured on a Murashige and Skoog basal salts medium supplemented with 53.6 μM 1-napthaleneacetic acid formed a compact, not friable type of callus that regenerated plantlets. Cormel slices and intact plantlets of three cultivars (‘Peter Pears’, ‘Rosa Supreme’, ‘Jenny Lee’) propagated through tissue culture formed a friable type of callus when cultured on Murashige and Skoog basal salts medium supplemented with 2,4-dichlorophenoxyacetic acid. This friable callus readily formed a cell suspension when the callus was placed in a liquid medium. Plants were regenerated from two-month-old suspension cell cultures of the commercial cultivar ‘Peter Pears’ after the suspension cells had been cultured on solid medium.     

In vitro induction, regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus

In the present paper attempts were made to induce chromosome doubling of ‘Meiwa’ kumquat (Fortunella crassifolia) protoplasts and ‘Frost’ navel orange (Citrus sinensis Osbeck) embryogenic callus via colchicine treatment. Colchicine decreased protoplast viability, delayed protoplast division and inhibited callus growth, indicating presence of toxicity to cells. Cell lines established from ‘Meiwa’ protoplasts treated with 0.01 and 0.1% colchicine for 8, 16 and 24 h at each concentration showed different responses when they were cultured on embryoid-induction medium. Flow cytometry (FCM) demonstrated that tetraploids were detected in cell lines and embryoids from all of the treatments, with the highest frequency being 19.23%. As for ‘Frost’, tetraploid cells were only detected when the callus was treated with 0.1% colchicine for either 4 or 8 days, from which plantlets were regenerated. FCM and chromosome counting confirmed them as true tetraploids. The diploid cells were more active in mitotic division during a 12-day culture and smaller in size than their tetraploid counterpart. Potential applications of the novel tetraploid germplasms obtained through in vitro chromosome doubling to citrus cultivar improvement are discussed.     

Micropropagation of fig (Ficus carica L.) ‘Roxo de Valinhos’ plants

Summary An in vitro protocol for Ficus carica cv. ‘Roxo de Valinhos’ was optimized. Nodal explants containing two buds were excised from field-grown mature plants, and transferred to different proliferation media consisting of combinations of distinct concentrations of activated charcoal with benzyladenine (BA), kinetin with gibberellic acid (GA₃), and WPM (woody plant medium) with kinetin. The regular strength of WPM in combination with 0.5 mgl⁻¹ kinetin was the best condition for shoot proliferation of Ficus carica ‘Roxo de Valinhos’ plants. The addition of activated charcoal in the medium completely inhibited shoot proliferation. The inclusion of BA in the medium induced excessive callus formation as well as small and vitrified shoots, while GA₃ induced excessive elongation associated with vitrification, chlorosis, and tip-burned shoots.     

Effects of different amino acids on somatic embryogenesis of strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch.)

This study was conducted to optimize different types and concentrations of amino acids on somatic embryogenesis induction, development and maturation of leaf explants in strawberry cultivars (‘Camarosa’, ‘Paros’ and ‘Kurdistan’). Calli derived from leaf sections were transferred onto MS medium with 1.0 mg/l 2,4-d + 0.5 mg/l BAP supplemented with 0.0, 50, 100, 150 or 200 mg/l concentrations of proline, alanine and glutamine. Stimulation of embryogenesis and embryo development was strictly dependent on the type and concentration of amino acid in the medium. Proline (100 mg/l) was much more effective than glutamine and alanine, on induction and development of somatic embryogenesis in all cultivars. Cultures grown on amino acid-free medium attained lower somatic embryos than cultures grown on amino acid treated medium. Low concentrations (50 mg/l) and high concentrations (200 mg/l) of amino acids tested were inefficient for embryogenesis induction as well as for somatic embryos development.     

Efficient and simple plant regeneration via organogenesis from leaf segment cultures of persimmon (Diospyros kaki thunb.)

Summary An efficient and simple plant regeneration system via organogenesis from leaf segments of persimmon (Diospyros kaki Thunb.) cultivars ‘Fuyu’ and ‘Nishimurawase’ has been developed. The regeneration capacity was influenced by the culture vessels, gelling agents, plant growth regulators, and light conditions. Leaf explants taken from in vitro shoots were cultured on a modified Murashige and Skoog medium (MS1/2N), for 16 wk without transfer to fresh medium. Adventious shoots appeared after 4 and 8 wk in culture of ‘Nishimurawase’ and ‘Fuyu’ tissues, respectively. The culture of leaf explants in Erlenmeyer flasks with medium containing 4 g l⁻¹ agar enhanced shoot formation in comparison to media with increased agar concentrations. Optimal shoot regeneration was obtained with 5 mg l⁻¹ (22.8 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) indole-3-butyric acid (IBA) for ‘Nishimurawase’, and 10 mg l⁻¹ (45.6 μM) zeatin and 0.1 mg l⁻¹ (0.05 μM) IBA for ‘Fuyn’. Shoot regeneration frequencies in both cultivars were 100%, and shoot numbers per explant reached up to 9.2 for ‘Nishimurawase’ and 2.2 for ‘Fuyu’. Dark incubation during the first 4–5 wk was the most effective condition to successfully influence shoot regeneration in both cultivars. While dark incubation was essential for adventitious shoot formation by ‘Fuyu’, it was only slightly beneficial to ‘Nishimurawase’. More than 80% of the regenerated shoots rooted within 4 wk on hormone-free MS1/2N demium after having been dipped for 30 s in 250 mg l⁻¹ (1.1. mM) IBA solution.     

Long-term study of somatic embryogenesis from anthers and ovaries of 12 grapevine (<Emphasis Type="Italic">Vitis</Emphasis> sp.) genotypes

Summary Anthers and ovaries of six grapevine cultivars (three Vitis vinifera L., two V × Labruscana L. H. Bailey, and one complex hybrid) were extracted from flower buds over 2 yr and cultured on three media reported to promote somatic embryogenesis in Vitis tissues. The highest percent embryogenesis from the hybrid ‘Chancellor’ and V. vinifera ‘Chardonnay’, ‘Merlot’, and ‘Pinot Noir’ occurred on medium C [Nitsch and Nitsch, 1969, basal medium with 3.0% (w/v) sucrose, 0.01% (w/v) inositol. 0.3% (w/v) Phytagel, 2.5 μM 2.4-dichlorophenoxyacetic acid, 2.5μM β-naphthoxyacetic acid, 5.0μM N-(2-chloro-4-pyridyl)-N′-phenylurea, and 0.05% (w/v) glutamine]. Regardless of the media, the labrusca cultivars ‘Concord’ and ‘Niagara’ produced soft non-embryogenic callus that was sometimes mixed with well-developed somatic embryos. Nine vinifera genotypes were further tested for several different years on medium C. Embryogenic cultures suitable for transformation were obtained from all genotypes in more than 1 yr. The average percent embryogenesis from ovaries was 7-fold higher than from anthers. There was significant annual variation in percent embryogenesis, demonstrating the need for media comparisons to be replicated for more than one season. Suspension cultures suitable for use in genetic transformation were initiated from ‘Chardonnay’, ‘Merlot,’ and ‘Pinot Noir’ pro-embryogenic masses. ‘Chardonnay’ suspension cultures plated and grown under conditions developed for recovery of plants after biolistic transformation yielded approximately 500 non-transformed embryos per plate after 4 mo. of culture, with 68.6% of the embryos converting to plants. This is the first reported protocol for embryogenesis from ‘Concord,’ ‘Cabernet Franc,’ and ‘Pinot Noir’ grapevines.     

Improved shoot organogenesis from hypocotyl segments of lingonberry (<Emphasis Type="Italic">Vaccinium vitis-idaea</Emphasis> L.)

Summary An improved protocol for shoot regeneration from hypocotyl segments of seedlings from open-pollinated seeds of lingonberry (Vaccinium vitis-idaea L.) cultivars, ‘Ida’, ‘Splendor’, and ‘Erntesegen’, and a native clone from Newfoundland was developed. The effect of thidiazuron (TDZ) on adventitious bud and shoot formation from apical, central, and basal segments of the hypocotyl was tested. Highly regenerative callus was obtained from hypocotyl segments on modified Murashige and Skoog (MMS) medium containing 5–10 μM TDZ. A maximum of 10 buds and 12 shoots per apical segment for seedlings of cultivar ‘Ida’ regenerated on MMS containing 10 μM TDZ. Callus and bud regeneration frequency, callus growth, and number of buds and shoots per regenerating explant depended not only on the specific segment of the hypocotyl, but also on parental genotype. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium containing 1–2 μM zeatin. The optimal concentration of sucrose for shoot elongation was 20 gl⁻¹. Shoots were rooted ex vitro on a 2 peat: 1 perlite (v/v) medium after dipping in 0.8% indole-3-butyric acid, and rooted plants acclimatized readily under greenhouse conditions.     

Effects of Auxins and Cytokinins on Embryo Formation from Root-derived Callus of Oncidium ‘Gower Ramsey’

In an attempt to optimize somatic embryo formation in Oncidium ‘Gower Ramsey’, the effects of five auxins (2,4-D, IAA, IBA, NAA and picloram) and five cytokinins (2iP, BA, kinetin, TDZ and zeatin), used alone, was tested in vitro using root-derived callus. In general, kinetin (0.5 and 2 mg l⁻¹) and zeatin (0.5 mg l⁻¹) were found to be more effective than other auxin and cytokinin treatments to induce somatic embryogenesis from root-derived callus.     

Exogenous ethylene influences flower opening of cut roses (<Emphasis Type="Italic">Rosa hybrida</Emphasis>) by regulating the genes encoding ethylene biosynthesis enzymes

The purpose of this paper is to investigate the differential responses of flower opening to ethylene in two cut rose cultivars, ‘Samantha’, whose opening process is promoted, and ‘Kardinal’, whose opening process is inhibited by ethylene. Ethylene production and 1-aminocyclopropane-1-carboxylate (ACC) synthase and oxidase activities were determined first. After ethylene treatment, ethylene production, ACC synthase (ACS) and ACC oxidase (ACO) activities in petals increased and peaked at the earlier stage (stage 3) in ‘Samantha’, and they were much more dramatically enhanced and peaked at the later stage (stage 4) in ‘Kardinal’ than control during vasing. cDNA fragments of three Rh-ACSs and one Rh-ACO genes were cloned and designated as Rh-ACS1, Rh-ACS2, Rh-ACS3 and Rh-ACO1 respectively. Northern blotting analysis revealed that, among three genes of ACS, ethylene-induced expression patterns of Rh-ACS3 gene corresponded to ACS activity and ethylene production in both cultivars. A more dramatic accumulation of Rh-ACS3 mRNA was induced by ethylene in ‘Kardinal’ than that of ‘Samantha’. As an ethylene action inhibitor, STS at concentration of 0.2 mmol/L generally inhibited the expression of Rh-ACSs and Rh-ACO in both cultivars, although it induced the expression of Rh-ACS3 transiently in ‘Kardinal’. Our results suggests that ‘Kardinal’ is more sensitive to ethylene than ‘Samantha’; and the changes of Rh-ACS3 expression caused by ethylene might be related to the acceleration of flower opening in ‘Samantha’ and the inhibition in ‘Kardinal’. Additional results indicated that three Rh-ACSs genes were differentially associated with flower opening and senescence as well as wounding.     

Shoot organogenesis in leaf explants of <Emphasis Type="Italic">Hydrangea macrophylla</Emphasis> ‘Hyd1’ and assessing genetic stability of regenerants using ISSR markers

For the first time, an in vitro regeneration protocol of Hydrangea macrophylla ‘Hyd1’ was developed. Effects of different plant growth regulators (PGRs) on shoot regeneration were investigated jointly with selecting optimal basal media and cefotaxime concentrations. The highest frequency of shoot organogenesis (100%) and mean number of shoots per explant (2.7) were found on Gamborg B5 basal medium supplemented with 2.25 mg/l 6-benzyladenine (BA), 0.1 mg/l Indole-3-butyric acid (IBA), 100 mg/l cefotaxime and 30 g/l sucrose solidified by 7 g/l agar. Regenerated shoots were rooted by culturing on perlite plus half strength liquid B5 basal medium with 0.5 mg/l NAA. Rooted plantlets were transplanted to the greenhouse with 100% survival rate. Genetic stability of 32 plantlets (one mother plant and 31 regenerants) was assessed by 44 ISSR markers. Out of 44 ISSR markers, ten markers produced clear, reproducible bands with a mean of 5.9 bands per marker. The in vitro regeneration protocol is potentially useful for the genetic transformation of Hydrangea macrophylla ‘Hyd1’.     

Somatic embryogenesis from mature zygotic embryos of commercial passionfruit (<Emphasis Type="Italic">Passiflora edulis</Emphasis> Sims) genotypes

Mature zygotic embryos of three genotypes of Passiflora edulis Sims, including ‘FB-100’, ‘FB-200’, and ‘FB-300’ were incubated on a Murashige and Skoog (MS) (1962) medium supplemented with different concentrations (18.1–114.8 μM) of 2,4-diclorophenoxyacetic acid (2,4-D) and 4.4 μM of 6-benzyladenine (BA). MS basal medium and MS with BA induced germination of P. edulis embryos. The highest frequencies of embryogenic calli were observed when explants were incubated on MS medium supplemented with 72.4 μM 2,4-D and 4.4 μM BA for ‘FB-200’, which showed the highest potential for embryogenic callus formation. Cytological and histological analyses of pro-embryogenic callus revealed two distinct cell types: thin-walled, small, isodiametric cells with large nuclei and dense cytoplasm, typical of intense metabolic activity; and elongated and vacuolated cells, with small nuclei and less dense cytoplasm. Differentiation of somatic embryos was promoted on MS medium supplemented with activated charcoal and indole-3-acetyl-l-aspartic acid (IAA-Asp) either with or without 2,4-D. However, no conversion of somatic embryos into plantlets was observed.     

Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy <Emphasis Type="Italic">Citrus</Emphasis> cultivars

Cytoplasmic male sterility (CMS) is known to be controlled by mitochondrial genome in higher plants including Satsuma mandarin (Citrus unshiu Marc.). Citrus symmetric fusion experiments often produce diploid cybrids possessing nuclear DNA from the mesophyll parent and mitochondrial DNA (mtDNA) from the embryogenic callus parent. Therefore, it is possible to transfer CMS from Satsuma mandarin as callus parent to seedy citrus cultivars as leaf one by somatic cybridization. Herein, symmetric fusion technique was adopted to create cybrids for potential seedlessness by transferring CMS from Citrus unshiu Marc. cv. Guoqing No. 1 (G1) to two traditional Chinese seedy citrus cultivars, ‘Shatian’ pummelo (C. grandis (L) Osbeck) and ‘Bingtang’ orange (C. sinensis (L) Osbeck). Flow cytometry analysis showed that 19 plants recovered from G1 + ‘Bingtang’ orange and 17 of 35 plants regenerated from G1 + ‘Shatian’ pummelo were diploid. The remaining plants from G1 + ‘Shatian’ pummelo were tetraploid. The diploid plants from the two combinations were confirmed as true cybrids by simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) analysis, with nuclear DNA from their corresponding leaf parent and mtDNA from their common suspension parent, G1 Satsuma mandarin. The remaining plants from G1 + ‘Shatian’ pummelo were identified as somatic hybrids with mtDNA from G1. The chloroplast simple sequence repeat (cp-SSR) analysis revealed somatic hybrid/cybrid plants from the two combinations in most cases possessed either of their parental chloroplast type, and two plants from G1 +‘Shatian’ pummelo and all embryoids analyzed from G1 + ‘Bingtang’ orange possessed chloroplast DNA (cpDNA) from both parents. These results demonstrated that we succeeded in introducing mtDNA from G1 Satsuma mandarin into the two target seedy citrus cultivars for potential seedlessness through symmetric fusion.     

Characterization of factors affecting plantlet regeneration from rice (Oryza sativa L.) callus

Various components of culture media were tested to characterize factors affecting plantlet regeneration from rice (Oryza sativa L.) callus. It was found that plantlet regeneration from rice callus was affected by concentrations of gelling agents, osmoticum, and the combination of hormones in the regeneration medium. High concentrations (4–6 g/l gellan gum, 10–16 g/l agar) of gelling agents promoted regeneration frequency. However, the total number of plantlets decreased with gellan gum concentrations above 4 g/l. Addition of sorbitol (15–75 g/l) promoted plantlet regeneration. However, the addition of mannitol was inhibitory and no regeneration was observed at concentrations above 30 g/l. This difference in the effects on regeneration suggests that sorbitol had another function besides as a osmoticum. High regeneration frequency was obtained with combinations of NAA (0.05–0.5 g/l) and kinetin (0.5–2 mg/l). However, higher concentrations (2 mg/l) of NAA are preferred to increase the total number of regenerated plantlets.     

Effect of 2,4-dichlorophenoxyacetic acid on callus induction and plant regeneration in anther culture of wheat (Triticum aestivum L.)

 Anthers from a doubled-haploid line of spring wheat (Triticum aestivum L.) cv. Pavon 76 were plated in liquid P-4 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at four concentrations (0.5, 1.0, 2.0, 4.0 mg/l) for 5, 10, 15, and 25 days before being transferred to another medium with the same or reduced 2,4-D concentrations for the remainder of the induction phase for a total of 45 days. Incubation with 0.5 mg/l 2,4-D for 45 days produced lower callus yield and plant regeneration, indicative of insufficient auxin for callus induction. Callus yield and regeneration frequencies were higher with 1.0 mg/l 2,4-D. With 2.0 or 4.0 mg/l 2,4-D, an induction period of 10 or 15 days was sufficient for initiation of callus development. The extended presence of 2–4 mg/l 2,4-D in the medium beyond the initiation phase was detrimental to plant regeneration. Thus optimal callus induction and plant regeneration could be obtained through manipulating the 2,4-D concentration and the duration of its presence in the induction medium. Received: 1 December 1997 / Revision received: 15 February 1999 / Accepted: 26 February 1999