The effect of auxin type and cytokinin concentration on callus induction and plant regeneration frequency from immature inflorescence segments of seashore paspalum (<Emphasis Type="Italic">Paspalum vaginatum</Emphasis> Swartz)

Seashore paspalum (Paspalum vaginatum Swartz) is a salt tolerant, fine textured turfgrass used on golf courses in coastal, tropical, and subtropical regions. A callus induction and plant regeneration protocol for this commercially important turfgrass species has been developed. Induction of highly regenerable callus with approximately 400 shoots per cultured immature inflorescence (1 cm in length) was achieved by culturing 0.2 cm segments on media with 3 mg l⁻¹ 3,6-dichloro-2-methoxybenzoic acid (dicamba) and 0.1 or 1.0 mg l⁻¹ benzylaminopurine (BA). A multifactorial experiment demonstrated the combination of 3 mg l⁻¹ dicamba and 1.0 mg l⁻¹ BA for induction of callus resulted in 12 times higher plant regeneration frequency compared to 3 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) alone or ten times higher plant regeneration frequency than the combination of 3 mg l⁻¹ 2,4-D and 1.0 mg l⁻¹ BA. These results are expected to support the development of a genetic transformation protocol for seashore paspalum.     

Transformation of <Emphasis Type="Italic">Liquidambar formosana</Emphasis> L. via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> using a mannose selection system and recovery of salt tolerant lines

Malaxis acuminata is a terrestrial orchid that grows in shady areas of semi-evergreen to shrubby forests. It is highly valued for its medicinal properties as dried pseudo-bulbs are important ingredients of several Ayurvedic preparations. In this study, adventitious shoot buds were induced from internodal explants of M. acuminata grown on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn), and thidiazuron (TDZ). Of the three cytokinins used, TDZ at 3 mg l⁻¹ induced the highest frequency (82%) of organogenic explants. However, all responding explants produced only a single adventitious shoot irrespective of the type and concentration of the cytokinin. Adding 0.5 mg l⁻¹ α naphthaleneacetic acid (NAA) to the medium enhanced adventitious shoot formation. In the presence of 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA, frequency of organogenesis was 96% with a mean number of 6.1 shoots per explant. Prolonged culture or subculture on the same medium did not promote further shoot production. However, transfer of these cultures to MS medium supplemented with 3 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA and various concentrations of different polyamines (PAs), including spermine, spermidine, and putrescine, significantly increased mean shoot number per explant. The highest frequency of shoot induction (100%) and mean shoot number per explant (14.6) was observed on MS medium with 3 mg l⁻¹ TDZ, 0.5 mg l⁻¹ NAA, and 0.4 mM spermidine. Regenerated shoots were excised and subcultured on an elongation medium consisting of MS medium with 3 mg l⁻¹ BA. Moreover, the highest frequency of rooting (96%) and mean number of roots per shoot (3.3) was observed on MS medium with 4 mg l⁻¹ indole-3-butyric acid (IBA) and 1.5 mg l⁻¹ activated charcoal (AC). Almost 90% of rooted shoots were successfully acclimatized and established ex vitro.     

Rapid multiplication of Polyscias filicifolia by secondary somatic embryogenesis

Efficient plant regeneration through somatic embryogenesis was achieved in Polyscias filicifolia. Embryogenic calluses were induced on Murashige and Skoog (MS) basal medium supplemented with 0.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l⁻¹ benzylaminopurine (BAP; type I callus) and on MS medium with 2.0 mg l⁻¹ 2,4-D and 0.01 mg l⁻¹ kinetin (type II callus) from leaf explants of a 2-yr-old plant. Primary somatic embryos (PSEs) developed after four passages of suspension culture established from embryogenic callus when cultured in liquid half-strength MS medium (1/2 MS) without growth regulators. PSEs in the cotyledonary stage were multiplied by adventitious embryogenesis. Single secondary somatic embryos (SSEs) or their clusters developed at the base of PSE hypocotyls and regenerated into plantlets in a one-step process on plant growth regulator-free 1/2 MS medium. Low sucrose concentration of 15 g l⁻¹ promoted development of normal SSEs. All SSEs regenerated into single, well-rooted plantlets on a Nitsch and Nitsch medium supplemented with 0.5 mg l⁻¹ kinetin, 0.1 mg l⁻¹ indole-3-butyric acid, and 10 mg l⁻¹ adenine sulfate. Subsequent two subculture cycles on the same medium were necessary to obtain plantlets sufficiency developed to allow successful transfer to the soil. Rooted plantlets were established in a peat mixture with 90% survival, with the plants showing normal morphological characteristics.     

Micropropagation of bromeliad <Emphasis Type="Italic">Aechmea fasciata</Emphasis> via floral organ segments and effects of acclimatization on plantlet growth

Although suckers and seedlings can be used for the propagation of bromeliads, the low number of propagules and cross-variation limit their uniformity and mass cultivation. In this study, high-efficiency shoot organogenesis and plant regeneration were achieved on callus derived from petal and ovary explants of Aechmea fasciata (Bromeliaceae). Calluses were induced on half-strength Murashige and Skoog inorganic salts (1/2MS) supplemented with 1.0–1.5 mg l⁻¹ 2,4-dichlorophenoxyacetic acid in combination with 1.0 or 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), and shoots regenerated after transfer to 1/2MS basal medium containing the combination of 1.0 mg l⁻¹ NAA + 0.5 mg l⁻¹ 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea. Those plantlets grown under a middle light intensity (50 μmol m⁻² s⁻¹) showed a dramatic increase in survival percentage (up to 95%) and the maximum number of newly developing roots. The plantlets that were transplanted onto pots were successfully grown in the greenhouse.     

Ethrel treatment enhanced isoflavonoids accumulation in cell suspension cultures of Pueraria tuberosa, a woody legume

The cell cultures of Pueraria tuberosa, a perennial leguminous lianas, were maintained in modified MS medium (KNO₃ 475 mg l⁻¹, thiamine 1 mg l⁻¹, biotin 1 mg l⁻¹, calcium pantothenate 1 mg l⁻¹) containing 0.1 mg l⁻¹ 2,4,5-trichloroacetic acid and 0.1 mg l⁻¹ kinetin. Isoflavonoids (puerarin, genistin, daidzein, genistein) accumulation in cell suspension cultures was increased by 14-fold to ~12 mg l⁻¹ after 48 h of adding 100 μM ethrel. Ethrel inhibitors (silver nitrate and silver thiosulfate) completely inhibited this effect in the presence of ethrel and isoflavonoids were not detected in the spent medium. The increase was dose dependent and can be explored to trigger high yield of isoflavonoids production.     

Effects of Rhamnolipids from Pseudomonas aeruginosa DS10-129 on Luminescent Bacteria: Toxicity and Modulation of Cadmium Bioavailability

In this study, the mixture of mono- and di-rhamnolipids produced by Pseudomonas aeruginosa DS10-129 was characterized for its toxicity and modulatory effects on Cd availability to different bacteria. Gram-negative naturally bioluminescent Vibrio fischeri and recombinant bioluminescent Pseudomonas fluorescens, P. aeruginosa, Escherichia coli, and Gram-positive Bacillus subtilis were used as model organisms. Rhamnolipids reduced the bioluminescence of these bacteria in less than a second of exposure even in relatively low concentrations (30-min EC₅₀ 45–167 mg l⁻¹). Toxicity of Cd to Gram-negative bacteria (30-min EC₅₀ values 0.16 mg l⁻¹ for E. coli, 0.96 mg l⁻¹ for P. fluorescens, and 4.4 mg l⁻¹ for V. fischeri) was remarkably (up to 10-fold) reduced in the presence of 50 mg l⁻¹ rhamnolipids. Interestingly, the toxicity of Cd to Gram-positive B. subtilis (30-min EC₅₀ value 0.49 mg l⁻¹) was not affected by rhamnolipids. Rhamnolipids had an effect on desorption of Cd from soil: 40 mg l⁻¹ rhamnolipids increased the water-extracted fraction of Cd twice compared with untreated control. However, this additionally desorbed fraction of Cd remained bound with rhamnolipids and was not available to bacteria. Hence, in carefully chosen concentrations (still effectively complexing heavy metals but not yet toxic to soil bacteria), rhamnolipids could be applied in remediation of polluted areas.     

Enhanced regeneration of haploid plantlets from microspores of <Emphasis Type="Italic">Brassica napus</Emphasis> L. using bleomycin,PCIB, and phytohormones

The effects of three periods of incubation (10, 20 and 30 min) at different levels of bleomycin (0, 0.1, 0.2, 0.3, 0.4 and 0.5 μg ml⁻¹), as well as three periods of exposure (12, 24 and 48 h) to different levels of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), including 1, 2, 3, 4 and 5 mg l⁻¹, on microspore embryogenesis of rapeseed cv. ‘Amica’ were investigated. Microspore embryogenesis was significantly enhanced following 20 min treatment with 0.2 μg ml⁻¹ bleomycin compared with untreated cultures. Highest embryo yield (163 embryos Petri dish⁻¹) was observed with 24 h treatment of 4 mg l⁻¹ PCIB. The highest percentage of secondary embryogenesis was observed on B₅ medium containing 0.15 mg l⁻¹ of gibberellic acid (GA₃) and 0.2 mg l⁻¹ 6-benzyladenine (BA) in 4–6 mm microspore-derived embryos (MDEs). Most callus formed on B₅ medium containing 0.15 mg l⁻¹ GA₃, 0.1 mg l⁻¹ BA and 0.1 mg l⁻¹ indole-3-acetic acid (IAA) when 4–6 mm embryos were used. Regeneration was highest on B₅ medium containing 0.05 mg l⁻¹ GA₃ or 0.1 mg l⁻¹ BA and 0.2 mg l⁻¹ IAA with 2–4 mm embryos. Microspore embryogenesis and plant regeneration could be improved by both bleomycin and PCIB when the appropriate MDE length and phytohormone level were selected.     

Plant regeneration from suspension culture of Iris germanica 1

In Iris germanica L., 'G1', 'Adorn' and 'Rococo', induction and proliferation of embryogenic calli were achieved by culture of leaf-base explants on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ 2,4-D, 1 mg l⁻¹ kinetin, 200 mg l⁻¹ casein hydrolysate, 250 mg l⁻¹ proline, 30 g l⁻¹ sucrose and 2.5 g l⁻¹ gellan gum. Among these cultivars, however, only in 'G1' could a suspension culture be established using a liquid N6 medium with 1 mg l⁻¹ 2,4-D, 1 mg l⁻¹ kinetin, 200 mg l⁻¹ casein hydrolysate, 250 mg l⁻¹ proline and 30 g l⁻¹ sucrose. Murashige and Skoog medium with 1 mg l⁻¹ gibberellic acid (GA₃), 30 g l⁻¹ sucrose and 2.5 g l⁻¹ gellan gum was suitable for somatic embryo formation from suspension cells. When the somatic embryos were transferred to solid, growth regulator-free MS medium and subcultured monthly, 36 shoots were obtained from 20 mg suspension cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Citric acid production from glycerol-containing waste of biodiesel industry by Yarrowia lipolytica in batch, repeated batch, and cell recycle regimes

Yarrowia lipolytica A-101-1.22 produces high citric acid (112 g l⁻¹) with a yield of 0.6 g g⁻¹ and a productivity of 0.71 g l⁻¹ h⁻¹ during batch cultivation in the medium with glycerol-containing waste of biodiesel industry. However, it was observed that the specific citric acid production rate, which was maximal at the beginning of the biosynthesis, gradually decreases in the late production phase and it makes continuation of the process over 100 h pointless. The cell recycle and the repeated batch regimes were performed as ways for prolongation of citric acid synthesis by yeast. Using cell recycle, the active citric acid biosynthesis (96–107 g l⁻¹) with a yield of 0.64 g g⁻¹ and a productivity of 1.42 g l⁻¹ h⁻¹ was prolongated up to 300 h. Repeated batch culture remained stable for over 1000 h; the RB variant of 30% feed every 3 days showed the best results: 124.2 g l^-1 citric acid with a yield of 0.77 g g^-1 and a productivity of 0.85 g l^-1 h^-1.     

Tissue culture and plant regeneration of blue grama grass, Bouteloua gracilis (H.B.K.) Lag. Ex Steud

Summary As a first step towards applying biotechnology to blue grama, Bouteloua gracilis (H. B. K.) Lag. ex Steud., we have developed a regenerable tissue culture system for this grass. Shoot apices were isolated from 3-d-old seedlings and cultured in 15 different growth regulator formulations combining 2,4-dichlorophenoxyacetic acid (2,4-D), Picloram (4-amino-3, 5,6-trichloropicolinic acid), N⁶-benzyladenine (BA) or adenine (6-aminopurine). The highest induction of organogenic callus was obtained with formulations containing 1 mg l⁻¹ (4.52 μM) 2,4-D plus 0.5 mg l⁻¹ (2.22 μM) BA. and 2 mg l⁻¹ (8.88 μM) BA plus 1 mg l⁻¹ (4.14 μM) Picloram with or without 40 mg l⁻¹ (296.08 μM) adenine. Lower frequencies of induction were obtained for embryogenic as compared to organogenic callus. The most efficient treatments for induction of embryogenic callus contained 2 mg l⁻¹ (9.05 μM) 2,4-D combined with 0.25 (1.11 μM) or 0.50 mg l⁻¹ (2.22 μM) BA, or 1 mg l⁻¹ (4.52 μM) 2,4-D with 0.50 mg l⁻¹ (2.22 μM) BA. Regeneration was achieved in hormonefree Murashige anmd Skoog (MS) medium, half-strength MS medium or MS medium plus 1 mg l⁻¹ (1.44 μM) gibberellic acid. The number of plantlets regenerated per 500 mg callus fresh weight on MS medium ranged from 9 for 2 mg l⁻¹ (9.05 μM) 2,4-D to 62.2 for induction medium containing 2 mg l⁻¹ (8,28 μM) Picloram, 1 mg l⁻¹ (4.44 μM) BA and 40 mg l⁻¹ (296.08 μM) adenine. Regnerated plants grown in soil under greenhouse conditions reached maturity and produced seeds.     

Agrobacterium tumefaciens-mediated transformation of Lesquerella fendleri L., a potential new oil crop with rich lesquerolic acid

A protocol was developed for regeneration and Agrobacterium-mediated genetic transformation of Lesquerella fendleri. Calli were first induced from hypocotyls and cotyledons on MS plus 0.5 mg l⁻¹ BA, 1 mg l⁻¹ NAA and 1 mg l⁻¹ 2,4-D, then co-cultivated for 2–3 days in darkness on MS supplemented with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹ NAA and 100 μmol l⁻¹As together with Agrobacterium tumefaciens strain EHA105/pCAMBIA1301 that harbored genes for uidA (GUS) and hygromycin resistance. Following co-cultivation, calli transfected by A. tumefaciens were transferred to MS with 0.5 mg l⁻¹ BA, 0.2 mg l⁻¹NAA, 500 mg l⁻¹ Cef and 10 mg l⁻¹ hygromycin and cultured for 10 days, then the hygromycin was increased to 20 mg l⁻¹ on the same medium. After 4 weeks the resistant regenerants were transferred to MS with 0.5 mg l⁻¹BA, 0.2 mg l⁻¹ NAA, 500 mg l⁻¹ Cef and 25 mg l⁻¹ hygromycin for further selections. Transgenic plants were confirmed by polymerase chain reaction analysis, GUS histochemical assay and genomic Southern blot hybridization. With this approach, the average regeneration frequency from transfected calli was 22.70%, and the number of regenerated shoots per callus was 6–13. Overall results described in this study demonstrate that Agrobacterium-mediated transformation is a promising approach for improvement of this Lesquerella species.     

Effects of plant growth regulators and sucrose on post harvest physiology,membrane stability and vase life of cut spikes of gladiolus

Effects of post-harvest application of two plant growth regulators viz., gibberellic acid (GA₃) and benzyl adenine (BA) with sucrose in the vase solution on cell membrane stability and vase life of gladiolus were investigated. The vase solution treatment combinations of GA₃ and BA with sucrose significantly increased the membrane stability index and enhanced the vase life as compared to the sucrose alone treatments or the controls. Vase solution treatment of GA₃ (50 mg l⁻¹), followed by BA (50 mg l⁻¹) with sucrose (50 g l⁻¹) significantly increased solution uptake, fresh weight and dry weight of cut spikes. The same treatments also enhanced the concentration of reducing and non-reducing sugars in gladioli petals 4 days after treatment (DAT). Cut spikes in vase solution enriched with 50 mg l⁻¹ GA₃ + 50 g l⁻¹ sucrose showed higher antioxidative enzyme activities of superoxide dismutase (SOD) and glutathione reductase (GR), lower lipoxygenase (LOX) activity and lipid peroxidation (measured as TBARS). Petal membrane stability index was also highest in cut spikes 6 DAT with 50 mg l⁻¹ GA₃ + 50 g l⁻¹ sucrose vase solution. Treatment of gladiolus cut spikes with 50 mg l⁻¹ GA₃ + 50 g l⁻¹ sucrose vase solution showed two fold increase in vase life and improved flower quality with a higher number of open flower per spike at any one time. These results suggest that post-harvest application of GA₃ (50 mg l⁻¹) with sucrose (50 g l⁻¹) maintains higher spike fresh and dry weight, improves anti-oxidative defence, stabilizes membrane integrity leading to a delay in petal cell death.     

Efficient plant regeneration from immature embryo cultures of <Emphasis Type="Italic">Jatropha curcas</Emphasis>, a biodiesel plant

Jatropha curcas L. (Physic nut) is a commercially important non-edible oil seed crop known for its use as an alternate source of biodiesel. In order to investigate the morphogenic potential of immature embryo, explants from four developmental stages were cultured on medium supplemented with combinations of auxins and cytokinins. It was found that the size of embryo is critical for the establishment of callus. Immature embryos (1.1–1.5 cm) obtained from the fruits 6 weeks after pollination showed a good response of morphogenic callus induction (85.7%) and subsequent plant regeneration (70%) with the maximum number of plantlets (4.7/explant) on Murashige and Skoog’s (MS) medium supplemented with IBA (0.5 mg l⁻¹) and BA (1.0 mg l⁻¹). The above medium when supplemented with growth adjuvants such as 100 mg l⁻¹ casein hydrolysate + 200 mg l⁻¹ l-glutamine + 8.0 mg l⁻¹ CuSO₄ resulted in an even higher frequency of callus induction (100%). Plant regeneration (90%) with the maximum number of plantlets (10/explant) was achieved on MS medium supplemented with 500 mg l⁻¹ polyvinyl pyrrolidone + 30 mg l⁻¹ citric acid + 1 mg l⁻¹ BA + 0.5 mg l⁻¹ Kn + 0.25 mg l⁻¹ IBA. It was observed that plantlet regeneration could occur either through organogenesis of morphogenic callus or via multiplication of pre-existing meristem in immature embryos. The age of immature embryos and addition of a combination of growth adjuvants to the culture medium appear to be critical for obtaining high regeneration rates. Well-developed shoots rooted on half-strength MS medium supplemented with 0.5 mg l⁻¹ IBA and 342 mg l⁻¹ trehalose. The rooted plants after acclimatization were successfully transferred to the field in different agro-climatic zones in India. This protocol has been successfully evaluated on five elite lines of J. curcas.     

Stimulation of menthol production in <Emphasis Type="Italic">Mentha piperita</Emphasis> cell culture

Plant cell culture provides an alternative means for producing secondary metabolites. In this study, experiments were carried out to study the impact of several parameters, independently and in combination, on the stimulation of menthol production in the cell suspension culture of Mentha piperita. Callus was obtained from leaf segments of in vitro grown plantlets on Murashige and Skoog (MS) medium supplemented with 0.2 mg l⁻¹of 2,4-dichlorophenoxy acetic acid to initiate cell suspension culture. This culture was maintained in half-strength MS medium supplemented with 0.2 mg l⁻¹of 2,4-dichlorophenoxy acetic acid at 15 d interval and used for further studies. Precursor feeding alone, i.e., menthone, at 35 μM concentration showed slightly improved productivity. γ-Cyclodextrin alone at 60 μM concentration and in combination with menthone feeding at 35 μM increased menthol yield up to 92 and 110 mg l⁻¹ in comparison to 77 mg l⁻¹ of control culture. Synergistic potentiation effect of menthone feeding at 35 μM and γ-cyclodextrin at 60 μM treatment followed by in situ adsorption with RP-8 also showed potential stimulation of menthol production in M. piperita cell culture. Fungal elicitor treatment showed enhanced production level up to 140.8 mg l⁻¹ in comparison to that of control. Further studies were carried out with the establishment of Agrobacterium tumefaciens (Ach5) gall-mediated calli, and consequently, cell suspension culture and results showed the significant enhancement of menthol yield up to 278 mg l⁻¹. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Guggulsterone production in cell suspension cultures of the guggul tree, <Emphasis Type="Italic">Commiphora wightii</Emphasis>, grown in shake-flasks and bioreactors

Cell suspension cultures of Commiphora wightii, grown in modified MS medium containing 2,4-dichlorophenoxyacetic acid (0.5 mg l⁻¹) and kinetin (0.25 mg l⁻¹), produced ∼5 μg guggulsterone g⁻¹ dry wt. In a 2 l stirred tank bioreactor, the biomass was 5.5 g l⁻¹ and total guggulsterone was 36 μg l⁻¹.     

Heterotrophic production of eicosapentaenoid acid by the diatom Nitzschia laevis: effects of silicate and glucose

The effects of silicate and glucose on growth and eicosapentaenoic acid (EPA) production by the diatom Nitzschia laevis were studied. By alternately altering the concentrations of silicate (2.7–64 mg l⁻¹) and glucose (1–40 g l⁻¹) in the medium, the highest cell dry weight (ca. 5.5 g l⁻¹) was obtained at 20 g l⁻¹ glucose and 32 mg l⁻¹ silicate, while the highest specific growth rate (ca. 0.65 day⁻¹) was obtained at a relatively low glucose concentration (5 g l⁻¹) and high silicate concentrations (32–64 mg l⁻¹). At glucose levels of 5 and 20 g l⁻¹, EPA content was higher with lower silicate concentrations (2.7 and 16 mg l⁻¹ silicate, respectively), while at a silicate level of 16 mg l⁻¹, higher glucose concentrations (20–40 g l⁻¹) facilitated EPA formation. The highest EPA yield (131 mg l⁻¹) was obtained at 20 g l⁻¹ glucose and 32 mg l⁻¹ silicate, while the highest EPA productivity (15.1 mg l⁻¹ day⁻¹) was obtained at 20 g l⁻¹ glucose and 64 mg l⁻¹ silicate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 218–224. Received 08 May 2000/ Accepted in revised form 21 July 2000     

Denitrification of a landfill leachate with high nitrate concentration in an anoxic rotating biological contactor

The denitrification performance of a lab-scale anoxic rotating biological contactor (RBC) using landfill leachate with high nitrate concentration was evaluated. Under a carbon to nitrogen ratio (C/N) of 2, the reactor achieved N-NO₃ ⁻ removal efficiencies above 95% for concentrations up to 100 mg N-NO₃ ⁻ l⁻¹. The highest observed denitrification rate was 55 mg N-NO₃ ⁻ l⁻¹ h⁻¹ (15 g N-NO₃ ⁻ m⁻² d⁻¹) at a nitrate concentration of 560 mg N-NO₃ ⁻ l⁻¹. Although the reactor has revealed a very good performance in terms of denitrification, effluent chemical oxygen demand (COD) concentrations were still high for direct discharge. The results obtained in a subsequent experiment at constant nitrate concentration (220 mg N-NO₃ ⁻ l⁻¹) and lower C/N ratios (1.2 and 1.5) evidenced that the organic matter present in the leachate was non-biodegradable. A phosphorus concentration of 10 mg P-PO₄ ³⁻ l⁻¹ promoted autotrophic denitrification, revealing the importance of phosphorus concentration on biological denitrification processes.     

<Emphasis Type="Italic">Aloe vera</Emphasis> transformation: the role of Amberlite XAD-4 resin and antioxidants during selection and regeneration

A system for genetic transformation and subsequent plant regeneration via indirect organogenesis from callus was developed for Aloe vera. Young seedlings served as primary explants. Callus cultures were established on Murashige and Skoog (1962) medium supplemented with 3 mg l⁻¹ benzylaminopurine and 2 mg l⁻¹ indole acetic acid. A protocol was developed to switch from the differentiated stage, using in vitro shoots or young regenerated plants, back to the de-differentiated stage of the callus and vice versa. Long-term maintenance of this callus paved the way for genetic manipulation of Aloe vera. Calluses were bombarded with a plasmid containing uidA and hpt genes, both under the control of the 35S promoter. Dithiothreitol and gibberellic acid were found to play a major role in reducing tissue necrosis following bombardment. Transformed shoots were regenerated under stepwise selection in hygromycin-containing liquid medium supplemented with different antioxidants. Amberlite XAD-4 resin was embedded into alginate beads and added to the selection medium. Amberlite was best for adsorbing different phenolic compounds and blocking explant necrosis. Shoot initiation occurred after transfer of the transformed cells to Murashige and Skoog medium supplemented with 2.0 mg l⁻¹ thidiazuron and 0.1 mg l⁻¹ indole butyric acid. Murashige and Skoog medium supplemented with 1 mg l⁻¹ zeatin riboside promoted shoot elongation. Rooting and plant development were obtained on Murashige and Skoog basal medium supplemented with 15 mg l⁻¹ hygromycin lacking growth regulators. The transgenic nature of the regenerated plants was verified by histochemical GUS assay and Southern blot hybridization.     

Characterization of Candida sp. NY7122, a novel pentose-fermenting soil yeast

Yeasts that ferment both hexose and pentose are important for cost-effective ethanol production. We found that the soil yeast strain NY7122 isolated from a blueberry field in Tsukuba (East Japan) could ferment both hexose and pentose (d-xylose and l-arabinose). NY7122 was closely related to Candida subhashii on the basis of the results of molecular identification using the sequence in the D1/D2 domains of 26S rDNA and 5.8S-internal transcribed spacer region. NY7122 produced at least 7.40 and 3.86 g l⁻¹ ethanol from 20 g l⁻¹ d-xylose and l-arabinose within 24 h. NY7122 could produce ethanol from pentose and hexose sugars at 37°C. The highest ethanol productivity of NY7122 was achieved under a low pH condition (pH 3.5). Fermentation of mixed sugars (50 g l⁻¹ glucose, 20 g l⁻¹ d-xylose, and 10 g l⁻¹ l-arabinose) resulted in a maximum ethanol concentration of 27.3 g l⁻¹ for the NY7122 strain versus 25.1 g l⁻¹ for Scheffersomyces stipitis. This is the first study to report that Candida sp. NY7122 from a soil environment could produce ethanol from both d-xylose and l-arabinose.     

Ethanol and xylitol production from glucose and xylose at high temperature by <Emphasis Type="Italic">Kluyveromyces</Emphasis> sp. IIPE453

A yeast strain Kluyveromyces sp. IIPE453 (MTCC 5314), isolated from soil samples collected from dumping sites of crushed sugarcane bagasse in Sugar Mill, showed growth and fermentation efficiency at high temperatures ranging from 45°C to 50°C. The yeast strain was able to use a wide range of substrates, such as glucose, xylose, mannose, galactose, arabinose, sucrose, and cellobiose, either for growth or fermentation to ethanol. The strain also showed xylitol production from xylose. In batch fermentation, the strain showed maximum ethanol concentration of 82 ± 0.5 g l⁻¹ (10.4% v/v) on initial glucose concentration of 200 g l⁻¹, and ethanol concentration of 1.75 ± 0.05 g l⁻¹ as well as xylitol concentration of 11.5 ± 0.4 g l⁻¹ on initial xylose concentration of 20 g l⁻¹ at 50°C. The strain was capable of simultaneously using glucose and xylose in a mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹, achieving maximum ethanol concentration of 38 ± 0.5 g l⁻¹ and xylitol concentration of 14.5 ± 0.2 g l⁻¹ in batch fermentation. High stability of the strain was observed in a continuous fermentation by feeding the mixture of glucose concentration of 75 g l⁻¹ and xylose concentration of 25 g l⁻¹ by recycling the cells, achieving maximum ethanol concentration of 30.8 ± 6.2 g l⁻¹ and xylitol concentration of 7.35 ± 3.3 g l⁻¹ with ethanol productivity of 3.1 ± 0.6 g l⁻¹ h⁻¹ and xylitol productivity of 0.75 ± 0.35 g l⁻¹ h⁻¹, respectively.