<Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>: colorful odyssey

Phaffia rhodozyma was isolated by Herman Phaff in the 1960s, during his pioneering studies of yeast ecology. Initially, the yeast was isolated from limited geographical regions, but isolates were subsequently obtained from Russia, Chile, Finland, and the United States. The biological diversity of the yeast is more extensive than originally envisioned by Phaff and his collaborators, and at least two species appear to exist, including the anamorph Phaffia rhodozyma and the teleomorph Xanthophyllomyces dendrorhous. The yeast has attracted considerable biotechnological interest because of its ability to synthesize the economically important carotenoid astaxanthin (3,3-dihydroxy-, -carotene-4,4-dione) as its major pigment. This property has stimulated research on the biology of the yeast as well as development of the yeast as an industrial microorganism for astaxanthin production by fermentation. Our laboratory has isolated several mutants of the yeast affected in carotenogenesis, giving colonies a vivid array of pigmentation. We have found that nutritional and environmental conditions regulate astaxanthin biosynthesis in the yeast, and have demonstrated that astaxanthin protects P. rhodozyma from damage by reactive oxygen species. We proposed in the 1970s that P. rhodozyma could serve as an economically important pigment source in animal diets including salmonids, lobsters, and the egg yolks of chickens and quail, in order to impart characteristic and desirable colors. Although P. rhodozyma/Xanthomyces dendrorhous has been studied by various researchers for nearly 30 years, it still attracts interest from yeast biologists and biotechnologists. There is a bright and colorful outlook for P. rhodozyma/X. dendrorhous from fundamental and applied research perspectives.     

Astaxanthin production by <Emphasis Type="Italic">Phaffia rhodozyma</Emphasis> and <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>: a comparative study

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) and Haematococcus pluvialis are known as the major prominent microorganisms able to synthesize astaxanthin natural pigment. Important research efforts have been made to determine optimal conditions for astaxanthin synthesis. When the focus is on astaxanthin production, the maximal reported value of 9.2 mg/g cell is obtained within H. pluvialis grown on BAR medium, under continuous illumination (345 μmol photon m⁻² s⁻¹) and without aeration. Whereas fermentation by mutated R1 yeast grown on coconut milk produced 1,850 μg/g yeast. However, when looking at astaxanthin productivity, the picture is slightly different. The figures obtained with P. rhodozyma are rather similar to those of H. pluvialis. Maximal reported values are 170 μg/g yeast per day with a wild yeast strain and 370 μg/g yeast per day with mutated R1 yeast. In the case of H. pluvialis, maximal values ranged from 290 to 428 μg/g cell per day depending on the media (BG-11 or BAR), light intensity (177 μmol photon m⁻² s⁻¹), aeration, etc. The main aim of this work was to examine how astaxanthin synthesis, by P. rhodozyma and H. pluvialis, could be compared. The study is based on previous works by the authors where pigment productions have been reported.     

Development of high frequency multiple shoot formation in Persian clover (<Emphasis Type="Italic">Trifolium resupinatum</Emphasis> L.)

An efficient and reliable micropropagation system for Persian clover (Trifolium resupinatum L.) was developed using different explants and media. Node, hypocotyl and cotyledonary node explants were cultured on Murashige and Skoog (MS) medium supplemented with combinations of either 6-benzyladenine (BA) and indole-3-butyric acid (IBA) or BA, Kinetin (KIN) and IBA. Direct multiple shoots developed within 6weeks in all explants in most media tested. The best shoot multiplication capacity was obtained from cotyledonary node explants on MS medium containing 7.1M BA and 1M IBA or 14.1M BA and 1M IBA. Elongated shoots were rooted on either MS medium alone or combination with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA) and -naphthaleneacetic acid (NAA). High rooting was achieved in half strength MS medium containing 8M IBA.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

An efficient <Emphasis Type="Italic">in vitro</Emphasis> method for mass propagation of <Emphasis Type="Italic">Tylophora indica</Emphasis>

A protocol of high frequency shoot organogenesis and plant establishment from stem derived callus has been developed for Tylophora indica (Burm. f.) Merrill. - an endangered medicinal plant. Callus was developed on Murashige and Skoog (MS) medium supplemented with 10 M 2,4,5-trichlorophenoxy acetic acid (2,4,5-T). Multiple shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (80 %) of shoot multiplication was achieved on MS medium containing 5.0 M kinetin. The developed shoots rooted best on half-strength MS medium supplemented with 0.5 M indole-3-butyric acid (IBA). The in vitro raised plantlets with well developed shoot and roots were acclimatized successfully and grown in greenhouse.     

<Emphasis Type="Italic">In vitro</Emphasis> induction of polyploidy in annatto (<Emphasis Type="Italic">Bixa orellana</Emphasis>)

The present work aims to establish a protocol for in vitro polyploidization using hypocotyl segments or cotyledonary nodes from in vitro grown annatto seedlings. The culture medium used to induce polyploidization was supplemented with MS salts, B5 vitamin complex, 100 mg l^– myo-inositol, 3% (w/v) sucrose, 2.28 M ZEA and 0.30 M IAA (hypocotyl segments) or 4.56 M ZEA (cotyledonary nodes), 0.8% (w/v) agar, and different concentrations of microtubule depolymerising agents, namely colchicine (0, 25, 250 and 1250 M) and oryzalin (0, 5, 15 and 30 M). To determine the optimum duration of either colchicine or oryzalin treatment for the induction of tetraploids, explants were treated for 15 or 30 days on regeneration medium. High frequencies of polyploidy in regenerated shoots from cotyledonary nodes were achieved in culture medium supplemented with 15 M oryzalin, for 15 days. Ploidy determination was based on chromosome counting in metaphasic cells from apical buds, and in the number of pairs of heterochromatic markers on the biggest chromosome, as visualized in interphasic nuclei, detection being easier in the latter. Among the characteristics evaluated, the measurements based on stomata length, width, area and frequency enabled greater discrimination between diploid and polyploid regenerated shoots.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Bambusa balcooa</Emphasis> Roxb.: Factors affecting changes of morphogenetic competence in the axillary buds

Axillary buds from field-grown culms of Bambusa balcooa were used as explants to induce multiple shoots in liquid Murashige and Skoogs (MS) medium supplemented with 11.25 M of 6-benzylaminopurine (BAP) and 4.5 M kinetin (Kn). A clump of at least 3 shoots was used for root induction in half strength MS medium with 1.0 M of 3-indolebutyric acid (IBA). Morphogenetic competence of the axillary buds varied widely in different months of two consecutive calendar years. The highest morphogenetic potentials were observed in October. The major problem encountered was presence of systemic fungal contaminants. Perhaps, rainfall positively contributed to induce morphogenetic competence. A moderately high phenolic content of the nodal explant was also detrimental for in vitro morphogenesis. The morphogenetic competence of B. balcooa correlated with the season in which the explants were excised from the natural stands. To the best of our knowledge this is the first report on in vitro regeneration of B. balcooa from mature field-grown axillary buds.     

<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated DNA transfer to<Emphasis Type="Italic"> Aesculu</Emphasis>s<Emphasis Type="Italic"> hippocastanum</Emphasis> L. and the regeneration of transformed plants

Hairy roots were induced from androgenic embryos of horse chestnut (Aesculus hippocastanum L.) by infection with Agrobacterium rhizogenes strain A4GUS. Single roots were selected according to their morphology in the absence of antibiotic or herbicide resistance markers. Seventy-one putative transformed hairy root lines from independent transformation events were established. Regeneration was induced in MS liquid medium supplemented with 30 6-benzylaminopurine (BA), and the regenerants were multiplied on MS solid medium containing 10 M BA. Following elongation on MS medium supplemented with 1 M BA and 500 mg/l polyvinylpyrrolidone, the shoots were subjected to a root-inducing treatment. Stable integration of TL-DNA within the horse chestnut genome was confirmed by Southern hybridization. The copy number of transgenes was estimated to be from two to four.Communicated by E.D. Earle     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Callus induction and <Emphasis Type="Italic">de novo</Emphasis> regeneration from callus in Guar (<Emphasis Type="Italic">Cyamopsis tetragonoloba</Emphasis>)

Guar (Cyamopsis tetragonoloba L. Taub) is a drought tolerant and multipurpose grain legume cash crop grown primarily under rainfed conditions in several countries. The effect of various growth regulators and their combinations on a variety of explants, namely the embryo, cotyledons, cotyledonary nodes, shoot tip and hypocotyle, has been studied and an efficient system for callus induction and regeneration from callus has been developed. It was established that Murashige and Skoogs culture medium containing 2,4-dichlorophenoxyacetic acid (10.0M) in combination with 6-benzylaminopurine (5.0M) with embryo or cotyledon explants is most suitable for induction of green and friable morphogenic callus, with a range of 82.5–95% of cultured explants responding to callus induction. Efficient de novo shoot regeneration was achieved by culturing the callus obtained on this medium on Murashige and Skoogs medium containing 1-naphthlenacetic acid (13.0M) in combination with 6-benzylaminopurine (5.0M) with a range of 82.1–88.4% of callus clumps producing 20–25 shoots. In vitro rooting of cultured shoots was obtained on half-salt concentration of Murashige and Skoogs culture medium supplied with indole-3-butyric acid (5.0M) on which 82–90% of cultured shoots produced healthy roots. The in vitro regenerated plants were grown to pod setting and subsequent maturity under greenhouse conditions.     

Cell volume distribution dynamics of <Emphasis Type="Italic">Chlorella</Emphasis><Emphasis Type="Italic">vulgaris</Emphasis> Beij. in batch cultures under continuous light

Cell volume distribution in Chlorella vulgaris cultures coming out of senescence was measured by flow cytometry every 6 h for 114 h in a full-factorial experiment with initial nitrate (420–4200 g NO₃-N l^–1), phosphate (9–186 g PO₄-P l^–1), and continuous light (50–330 E m^–2 s^–1) as treatments. The maxima in median and median absolute deviation (MAD) of cell volume were achieved within 6 h of each other and their timing was not affected by any treatment. Population specific growth rate during the first 66 h calculated from volume distribution changes was significantly affected by light treatment only (p=0.002).Revisions requested 4 November 2004; Revisions received 17 January 2005     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration of <Emphasis Type="Italic">Alnus acuminata</Emphasis> H.B.K. ssp. <Emphasis Type="Italic">acuminata</Emphasis> and its root nodulation by <Emphasis Type="Italic">Frankia</Emphasis>

A procedure for in vitro plant regeneration of Alnus acuminata from epicotyls with cotyledonary buds was developed using different media formulations with different growth regulators and carbon sources. The development of multiple buds on explants at the initiation step was obtained with MS at 1/2 strength with either 1 or 2M of BAP but not without it. Multiplication gave up to 15 elongating shoots by explant, the best medium being MS supplemented with vitamins from B5 medium, 1M of BAP and 87mM sucrose. Rooting of about 88% occurred in the medium MS with 83 mM sucrose and 1M IBA. Alnus acuminata did not developed well on WPM. Roots of in vitro propagated plants were nodulated by Alnus-infective Frankia. The root nodules show a typical alder root nodule anatomy and differentiation pattern and effectively fixed nitrogen. Rhamnaceae-infective Frankia did not nodulate in vitro cultivated Alnus acuminata suggesting that symbiotic recognition was not altered by in vitro regeneration of the plant.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Tissue culture propagation of Mongolian cherry (<Emphasis Type="Italic">Prunus fruticosa</Emphasis>) and Nanking cherry (<Emphasis Type="Italic">Prunus tomentosa</Emphasis>)

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 M indole-3-butyric acid (IBA) and either 4.44 or 8.88 M 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 M BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 M thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 M) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.     

Development of adventitious shoots from <Emphasis Type="Italic">in vitro</Emphasis> grown <Emphasis Type="Italic">Cydonia oblonga</Emphasis> leaves as influenced by different cytokinins and treatment duration

The effects of three cytokinins, kinetin 4.5 M (Kin), 6-benzylaminopurine 4.5 M (BA) and N-phenyl-N1,2,3- thiadiazol-5-yl-urea 4.5 M (TDZ), and the effects of different treatment duration on the regeneration of adventitious shoots from quince (Cydonia oblonga Mill.) leaves were studied. In a first experiment, leaves treated with Kin for 0, 8, 16 and 24 d were transferred to BA or TDZ-containing growth medium. In a second experiment TDZ applied for 0, 4, 8, 12, 16 and 24 d was followed by BA. All treatments included 0.5 M -naphthaleneacetic acid (NAA). In the sequence Kin-BA, the production of adventitious shoots decreased and reddish-coloured nodular structures (RNS) of meristematic appearance increased with increasing duration of Kin treatment, while somatic embryo formation was optimal at 8 d. In the Kin-TDZ sequence, shoot production was initially pronounced, but it declined with increasing duration of the Kin treatment, while the number of roots, somatic embryos and RNS increased. TDZ-BA treatments induced marked shoot production, which gradually increased with increasing duration of TDZ treatment. The presence of TDZ and a long treatment duration appeared to be very important factors in inducing caulogenesis. Kin appeared to be effective in shoot induction but not in shoot development; the results of this work demonstrate that RNS were adventitious shoots blocked at an early developmental stage on account of insufficient cytokinin activity. BA was less effective than TDZ in inducing shoot regeneration. Finally, both Kin and BA applied after 2,4-D treatment promoted somatic embryo induction.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Perilla frutescens</Emphasis> from hypocotyl and cotyledon explants

Organogenetic buds were induced from hypocotyl and cotyledon explants of oil crop Perilla frutescens in Murashige and Skoog (MS) medium supplemented with 5.7 M indole-3-acetic acid (IAA) and 8.9 – 13.3 M 6-benzylaminopurine (BA). Shoots were rooted on MS medium with 2.9 M IAA and 1.4 M gibberellic acid (GA3) and the regenerated plants flowered and set seeds normally.     

Development of a shoot regeneration protocol for genetic transformation in <Emphasis Type="Italic">Pelargonium zonale</Emphasis> and <Emphasis Type="Italic">Pelargonium peltatum</Emphasis> hybrids

The attempts of this investigation were to develop a system for plant regeneration from explants of adult plants and its use for genetic transformation of important commercial Pelargonium zonale hybrid and P. peltatum hybrid cultivars. To this aim, leaf blade and petiole explants of eight cultivars were cultured on modified MS (Murashige and Skoog, 1962) medium with two concentrations of TDZ, BA, and zeatin (5 and 20 M). Petiole explants showed a higher regeneration response than leaf blade explants and TDZ was the most effective cytokinin. Petioles of 16 cultivars were incubated on medium containing 5, 10, 15, and 20 M TDZ, respectively, in order to identify the most effective TDZ concentration. For the majority of genotypes 10 M TDZ resulted in the best regeneration response. Cefotaxim at 500 mg l ^–1 had no effect on shoot regeneration and did not show interaction with glufosinate. For selection of transgenic cells, a concentration of 2.5 M glufosinate was shown to be appropriate. LBA4404 and EHA101 Agrobacterium strains carrying pIBGUS vector with pat gene as selectable marker gene and GUS gene as reporter gene were compared in transformation studies. With regard to GUS expression in petiole explants 16 days after coculture, better results were obtained with EHA 101 than with LBA 4404.     

Bioavailability of zinc in the sediment to the estuarine amphipod <Emphasis Type="Italic">Grandidierella</Emphasis><Emphasis Type="Italic">japonica</Emphasis>

The utility of interstitial water concentrations of metals and simultaneously extracted metals/acid-volatile sulfide differences (SEM–AVS) in two seasons were investigated to explain the biological availability of zinc in sediments to benthic organisms exposed in the laboratory. The amphipod Grandidierella japonica was exposed, in 10-day acute toxicity tests, to clean sediment spiked with zinc to obtain nominal treatments ranging from 0.25 to 74.4 mol g^–1 dry weight with respect to the molar difference between SEM_Zn and AVS. When the molar difference between SEM_Zn and AVS (i.e., SEM–AVS) was <0 mol g^–1, the concentration of zinc in the sediment interstitial water was low and few adverse effects were observed for any of the biological endpoints measured. Conversely, when SEM_Zn–AVS exceeded 0 mol g^–1, the concentration of zinc in the interstitial water and amphipod mortality increased. These data compare favorably with observations made in short-term exposures and thus support the use of AVS as a normalization phase for predicting toxicity in metal-contaminated sediments in different season.     

Fumonisin production by <Emphasis Type="Italic">Fusarium</Emphasis> species isolated from freshly harvested corn in Iran

Fifty-one strains of Fusarium verticillioides and F. proliferatum isolated from corn collected from four different geographic areas in Iran, namely Fars, Khuzestan, Kermanshah and Mazandaran (an endemic oesophageal cancer (OC) area) were evaluated for their ability to produce fumonisins B₁ (FB₁), B₂ (FB₂) and B₃ (FB₃) in corn culture. Fumonisin levels were determined by high-performance liquid chromatography. All tested strains of F. verticillioides and F. proliferatumproduced fumonisins within a wide range of concentrations, 197–9661 g/g, 18–1974 g/g, and 21–1725 g/g for FB₁, FB₂, and FB₃, respectively. The highest mean concentrations of FB₁, FB₂, and FB₃ were 3897, 806 and 827 g/g, respectively. Overall, 61% of the F. verticillioides and F. proliferatum strains produced higher levels of FB₃ than FB₂. The mean ratios of FB₁:FB₂, FB₁:FB₃ and FB₁:total fumonisins were 8, 7 and 0.7 for F. verticillioides and 5.7, 10.7 and 0.7 for F. proliferatum, respectively. Significant differences in some of the meteorological data (rainfall, relative humidity and minimum temperature) from the four provinces were observed. Fumonisin levels produced by F. verticillioides strains isolated from Khuzestan province (tropical zone) were significantly (P < 0.01) higher than the other three provinces. This is the first report of the fumonisin-producing ability of F.verticillioides and F. proliferatum strains isolated from corn harvested from different geographic areas in Iran.     

Silver nitrate and aminoethoxyvinylglycine affect <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated apple transformation

In Fuji, the production of ethylene was increased with the addition of AgNO₃ and inhibited with the addition of 10 M aminoethoxyvinylglycine (AVG). The addition of 80 M AgNO₃ to transformed explants of Fuji cultured on selection medium resulted in increased ethylene production (20 l l^–1) at 3 weeks. Under examining the effect of AgNO₃ in Fuji, the 40 M AgNO₃ showed with higher 33.8% and 6.5% in the efficiency of regeneration and transformation. However, ethylene production in Gala explants treated with 10M AgNO₃ (3 l l^–1) decreased after 2 weeks compared with the control (5 l l^–1). Although the regeneration efficiency of Gala with 10 M AgNO₃ was higher (41.1%) than the control (20.1%), there was no significant difference in the transformation efficiency at the same concentration. Shoot regeneration of Fuji and Gala was completely inhibited with 10 M AVG. These results suggest that the addition of AgNO₃ affects the efficiency of Agrobacterium-mediated gene transfer in Fuji.Eun Soo Seong, Ill Min Chung- These two Authors Contributed equally to this work