<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from the immature seeds of <Emphasis Type="Italic">Cymbidium faberi</Emphasis>

An in vitro plant regeneration protocol of Cymbidium faberi from immature seeds was established. The immature seeds of 50 days old started to form rhizomes 4 months after they were cultured on hormone free medium. The rhizomes multiplied 5 times when subcultured on the medium containing 1.0 mg l^–1 -naphthalene acetic acid (NAA) for 40 days and more than 90% of the rhizomes initiated shoots within 60 days on the media containing 0.5 or 1.0 mg l^–1 NAA plus 2.0 or 5.0 mg l^–1N⁶-benzylaminopurine (BA). Plantlets were regenerated when the shoots were planted on the basal medium amended with 1 g l^–1 activated charcoal for 50 days and the plantlets grew normally after transplanting.     

<Emphasis Type="Italic">In vitro</Emphasis> flowering of <Emphasis Type="Italic">Perilla frutescens</Emphasis>

This study investigated the factors affecting in vitro flowering of Perilla frutescens. The shoots regenerated from cotyledonary and hypocotyl explants cultured on Murashige and Skoog (MS) medium supplemented with benzyladenine (BA) and indole-3-acetic acid, each at 0.5 mg l⁻¹, were excised and transferred to MS medium containing 30 g l⁻¹ of sucrose, 8.25 g l⁻¹ of ammonium nitrate, and 1.0 mg l⁻¹ of BA. After 40 d of culture, 86.2% of shoots flowered and most of which self-fertilized in vitro and produced mature fruits with viable seeds. These seeds were germinated and plants were grown to maturity and flowered in soil under greenhouse conditions. The in vitro flowering system reported in this study may facilitate rapid breeding of P. frutescens and offers a model system for studying the physiological mechanism of flowering.     

Direct organogenesis from leaf explants of <Emphasis Type="Italic">Stevia rebaudiana</Emphasis> and cultivation in bioreactor

Shoot buds were induced directly on either side of midrib from adaxial surface of immature leaf explants in Stevia rebaudiana Bertoni five weeks after culturing in Murashige and Skoog’s nutrient medium supplemented with 8.88 μM of N ⁶-benzylaminopurine and kinetin ranging from 4.65 to 6.98 μM. Immature leaves of 0.6 to 1 cm were found to produce best response (93 %) with a highest number of 4.93 shoot buds per explant. For elongation of regenerated shoot buds, MS medium supplemented with 30 g dm⁻³ sucrose and indole-3-butyric acid (IBA) ranging from 4.92 to 7.38 μM were found most suitable. The medium was further modified to suit bioreactor cultivation of regenerated shoots wherein the use of two-fold MS salts and 60 g dm⁻³ sucrose resulted in a high biomass yield of 50.68 g dm⁻³ (m/v) accounting for about 590 micro-cuttings in three weeks. Best rooting of micro-cuttings occurred in half strength MS medium supplemented with IBA ranging from 4.92 to 7.38 μM, 15 g dm⁻³ sucrose and gelled with 0.8 % agar. Rooted plants were successfully established in substrate containing sand, Vermicompost and garden soil in equal proportions and grown in greenhouse. This is the first report on direct shoot regeneration from Stevia leaves.     

Enhancing yield of <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by controlling NH<Subscript>4</Subscript><Superscript>+</Superscript> concentration

Yield of S-adenosylmethionine was improved significantly in recombinant Pichia pastoris by controlling NH₄ ⁺ concentration. The highest production rate was 0.248 g/L h when NH₄ ⁺ concentration was 450 mmol/L and no repression of cell growth was observed. Within very short induction time (47 h), 11.63 g/L SAM was obtained in a 3.7 L bioreactor.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of the chinese medicinal plant, <Emphasis Type="Italic">Dendrobium candidum</Emphasis> wall. ex lindl., from axenic nodal segments

Summary Dendrobium candidum Wall. Ex Lindl. is an important species used in the formulation of Shih-hu, a Chinese traditional medicine. An efficient protocol for in vitro propagation of D. candidum using the axenic nodal segments of the shoots, originated from the in vitro germinated seedlings, was developed. The seeds from 120-d-old capsules after pollination were first germinated on half-strength Murashige and Skoog (MS) basal medium supplemented with 30 g l⁻¹ sucrose. After 4 mo., the seedlings were subcultured on a similar medium supplemented with 1 ml l⁻¹ HYPONeX, 80 g l⁻¹ potato homogenate and 2 g l⁻¹ activated charcoal for further growth. Axenic nodal segments excised from 9-mo.-old seedlings were cultured on the medium in the presence of 2 mg l⁻¹ benzyladenine (BA) and 0.1 mg l⁻¹ naphthaleneacetic acid (NAA). After 75 d, 73.2% of the explants gave rise to buds/shoots. The elongated shoots were rooted on the medium containing 0.2 mg l⁻¹ NAA and the plantlets were successfully acclimatized in soil.     

Direct shoot regeneration from nodal explants of <Emphasis Type="Italic">Sida cordifolia</Emphasis> Linn

A method was developed to initiate multiple shoots from mature nodal explants of Sida cordifolia Linn. High frequency of regeneration was achieved on Murashige and Skoog (MS) medium supplemented with 2.0 mg l⁻¹ 6-benzylaminopurine, 0.5 mg l⁻¹ α-naphthalene acidic acid, 1.0 mg l⁻¹ adenine sulfate, and 10% (v/v) coconut milk. Multiple shoots were initiated within 21 d and the above media was capable of inducing the formation of more than 20 shoots from each explant. Regenerated shoots were successfully rooted on half-strength MS medium supplemented with 2.0 mg l⁻¹ indole-3-butyric acid and 3% (w/v) sucrose. Rooted plantlets were established in soil. The regenerated plantlets showed no morphological differences from the parent material. This protocol could be useful for germplasm conservation, cultivation, and genetic improvement of S. cordifolia.     

Expansion of <Emphasis Type="Italic">Phragmites australis</Emphasis> (Cav.) Trin. ex Steud. (Common Reed) into <Emphasis Type="Italic">Typha</Emphasis> spp. (Cattail) Wetlands in Northwestern Indiana,USA

Expansion of Phragmites australis (Cav.) Trin. ex Steud. (common reed) into stands of Typha spp. (cattail; Typha australis L. and T. x glauca) is common in the wetlands of northwestern Indiana (USA). To understand this phenomenon better, we investigated the production of shoot sprouts and proportional allocation of biomass as well as a potential role for the water table in the relative dominance of each species. The reduction in sprouts from rhizomes upon vegetative expansion of Phragmites appeared to be the most likely process causing the decline of Typha. The latter had a shoot density of 39/m² in plots without Phragmites, but this dropped to 13 shoots m⁻² in plots that had been invaded by Phramites. Such a decline was likely caused by reduced reserves; e.g., the belowground biomass of Typha decreased from 11.3 g m⁻² without Phragmites to 8.1 g m⁻² with Phragmites. The latter also reduced its belowground biomass but not its shoot density in the presence of Typha. The mean weight of Phragmites shoots was 2.9 g, and nearly all produced inflorescences. Meanwhile, Typha failed to develop spadices despite its shoots having a greater biomass (7 g). This suggests that Phragmites is more efficient than Typha in shoot growth. Springtime flooding appeared to promote the sprout of Typha shoots from shallow rhizomes (≈18 cm below the soil surface), whereas the shoot density of Phragmites showed no correlation with water level in that season. Deep-rooted Phragmites (≈39 cm) occurred on both high and low water-table sites, whereas the shallow-rooted Typha was limited to only the former. Phragmites will likely continue its expansion, by vegetative sprouts from rhizomes, into Typha wetlands.     

Production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate) P(3HB-<Emphasis Type="Italic">co</Emphasis>-3HV) with a broad range of 3HV content at high yields by <Emphasis Type="Italic">Burkholderia sacchari</Emphasis> IPT 189

The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from sucrose and propionic acid by Burkholderia sacchari IPT 189 was studied using a two-stage bioreactor process. In the first stage, this bacterium was cultivated in a balanced culture medium until sucrose exhaustion. In the second stage, a solution containing sucrose and propionic acid as carbon source was fed to the bioreactor at various sucrose/propionic acid (s/p) ratios at a constant specific flow rate. Copolymers with 3HV content ranging from 40 down to 6.5 (mol%) were obtained with 3HV yield from propionic acid (Y _3HV/prop) increasing from 1.10 to 1.34 g g⁻¹. Copolymer productivity of 1 g l⁻¹ h⁻¹ was obtained with polymer biomass content rising up to 60% by increasing a specific flow rate at a constant s/p ratio. Increasing values of 3HV content were obtained by varying the s/p ratios. A simulation of production costs considering Y _3HV/prop obtained in the present work indicated that a reduction of up to 73% can be reached, approximating US$ 1.00 per kg which is closer to the value to produce P3HB from sucrose (US$ 0.75 per kg).     

A simple method for mass propagation of Spathiphyllum cannifolium using an airlift bioreactor

Summary A method for the micropropagation of Spathiphyllum cannifolium is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. The proliferation responses were significantly influenced by the cytokinin type and concentrations. Supplementation of the medium with benzyladenine (BA; 4.44–13.32 μM) increased the shoot proliferation rate significantly as compared to other treatments. When cytokinins were used with auxin (indole-3-butyric acid, IBA and naphthalene acetic acid. NAA), the number of shoots per explant increased in comparison with treatments with BA alone. The largest number of shoots, 9.3 per explant, was obtained with 13.32 μM BA and 4.9 μM IBA. Different MS medium strengths and sucrose concentrations were used with the aim to stimulate in vitro shoot proliferation. Full MS medium with 30 gl⁻¹ sucrose was found to be suitable for shoot tip culture of Spathiphyllum. Comparative studies between gelled medium and bioreactor culture [continuous immersion (with or without net) and temporary immersion in liquid media using ebb and flood] revealed that shoot multiplication and growth were more efficient in continuous immersion (with net) bioreactor with low cytokinin-supplemented media. Plantlets from the bioreactor were cultured hydroponically for 30 d and 100% of plants were rooted and acelimatized successfully. Rapid and efficient multiplication rate in bioreactor and successful transfer to greenhouse makes this protocol suitable for large-scale multiplication of this important foliage plant.     

High efficiency degradation of tetrahydrofuran (THF) using a membrane bioreactor: identification of THF-degrading cultures of<Emphasis Type="Italic"> Pseudonocardia</Emphasis> sp. strain M1 and<Emphasis Type="Italic"> Rhodococcus ruber</Emphasis> isolate M2

A mixed microbial culture capable of growing aerobically on tetrahydrofuran (THF) as a sole carbon and energy source was used as the inoculum in a 10 l working volume membrane bioreactor. Following start-up, the reactor was operated in batch mode for 24 h and then switched to continuous feed with 100% biomass recycle. On average, greater than 96% of THF fed to the reactor was removed during the 8-month study. THF loading rates ranged from 0.62 to 9.07 g l^–1 day^–1 with a hydraulic retention time of 24 h. THF concentrations as high as 800 mg/l were tolerated by the culture. Biomass production averaged 0.28 kg total suspended solids/kg chemical oxygen demand removed, i.e., comparable to a conventional wastewater treatment process. Periodic batch wasting resulted in a solids retention time of 7–14 days. Reactor biomass typically ranged from 4 to 10 g/l volatile suspended solids and the effluent contained no solids. Pure THF-degrading cultures were isolated from the mixed culture based on morphological characteristics, Gram-staining and THF degradation. Based on 16S rDNA analysis the isolates were identified as Pseudonocardia sp. M1 and Rhodococcus ruber M2.     

Phytotoxic, clastogenic and bioaccumulation effects of the environmental endocrine disruptor bisphenol A in various crops grown hydroponically

The effects of the endocrine disruptor bisphenol A (BPA) at concentrations of 10 and 50 mg l⁻¹ were evaluated on the germination and morphology, micronuclei (MN) content in root tip cells and BPA bioaccumulation of hydroponic seedlings of broad bean (Vicia faba L.), tomato (Lycopersicon esculentum Mill.), durum wheat (Triticum durum Desf.) and lettuce (Lactuca sativa L.) after 6 and 21 days of growth. In general, BPA at any dose used did not inhibit germination and early growth (6 days) of seedlings of the species examined, with the exception of primary root length of tomato which decreased at the higher BPA dose. In contrast, an evident phytotoxicity was induced by BPA in all species after 21 days of growth with evident morphological anomalies and significant reductions of the lengths and fresh and dry weights of shoots and roots of seedlings. With respect to the nutrient medium without seedlings, BPA concentration decreased markedly during the growth period in the presence of broad bean and tomato seedlings, and limitedly in the presence of durum wheat and, especially, lettuce. Further, the presence of BPA measured in roots and shoots of broad bean and tomato after 21-day growth indicated that bioaccumulation of BPA had occurred. The number of MN in broad bean and durum wheat root tip cells increased markedly by treatment with BPA at both concentrations, thus suggesting a potential clastogenic activity of BPA in these species.     

Carbon autonomy of reproductive shoots of Siberian alder (<Emphasis Type="Italic">Alnus hirsuta</Emphasis> var.<Emphasis Type="Italic"> sibirica</Emphasis>)

Carbon autonomy of current-year shoots in flowering, and of current-year shoots plus 1-year-old shoots (1-year-old shoot system) in fruiting of Siberian alder (Alnus hirsuta var. sibirica) was investigated using a stable isotope of carbon, ¹³C. The current-year shoot and 1-year-old shoot systems were fed ¹³CO₂ and the atom% excess of ¹³C in flowers and fruits was determined. The majority of photosynthate allocated to flower buds was originally assimilated in the leaves of the flowering current-year shoots. Of all the current-year shoots on fruiting 1-year-old shoots, only those nearest to the fruits allocated the assimilated photosynthate to fruit maturation. These results indicate that the current-year shoots and 1-year-old shoot systems are carbon-autonomous units for producing flowers and maturing fruits, respectively.     

The effect of co-cultivation and selection parameters on <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Chinese soybean varieties

In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l⁻¹) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone. The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with 3 mg l⁻¹ hygromycin for 10 days, 5 mg l⁻¹ hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l⁻¹ hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression and inheritance of the transgenes were confirmed by molecular and genetic analysis. T₁ plants were analyzed and transmission of transgenes to the T₁ generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation.     

Micropropagation of zedoary (<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Roscoe) – a valuable medicinal plant

Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l^–1 sucrose and 5 g l^–1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l^–1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l^–1 BA, and 0.5 mg l^–1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l^–1 sucrose and 8 g l^–1 agar, supplemented with 20 (v/v) CW and 2 mg l^–1 NAA. More than 95 of the rooted plants were established in pots after hardening.     

Production of eleutherosides in in vitro regenerated embryos and plantlets of <Emphasis Type="BoldItalic">Eleutherococcus chiisanensis</Emphasis>

High frequency somatic embryogenesis of Eleutheorcoccus chiisanensis was achieved through suspension culture of embryogenic cells in hormone-free Murashige and Skoog liquid medium supplemented with 30 g sucrose l⁻¹. Cotyledonary somatic embryos were germinated and converted into plantlets using 20 μM gibberellic acid which were then grown in a 10 l airlift bioreactor. HPLC analysis revealed the accumulation of eleutheroside B, E and E1 in the embryos and plantlets. Thus mass production of embryos and plantlets of E. chiisanensis can be achieved in liquid cultures and the biomass produced may become an alternative source of eleutherosides.     

Bioreactor for solid-state cultivation of <Emphasis Type="Italic">Phlebiopsis gigantea</Emphasis>

Phlebiopsis gigantea fungus used in biological control of root rot is currently cultivated commercially in disposable, sterilizable plastic bags. A novel packed bed bioreactor was designed for cultivating P. gigantea and compared to the plastic bag method and to a tray bioreactor. The spore viability of 5.4 × 10⁶ c.f.u./g obtained with the packed bed bioreactor was of the same order of magnitude as the viabilities obtained with the other cultivation methods. Furthermore, the packed bed bioreactor was less time and space consuming and easier to operate than the tray bioreactor.     

Continuous production of citric acid from dairy wastewater using immobilized<Emphasis Type="Italic">Aspergillus niger</Emphasis> ATCC 9142

The continuous production of citric acid from dairy wastewater was investigated using calcium-alginate immobilizedAspergillus niger ATCC 9142. The citric acid productivity and yield were strongly affected by the culture conditions. The optimal pH, temperature, and dilution rate were 3.0, 30°C, and 0.025 h⁻¹, respectively. Under optimal culture conditions, the maximum productivity, concentration, and yield of citric acid produced by the calcium-alginate immobilizedAspergillus niger were 160 mg L⁻¹ h⁻¹, 4.5 g/L, and 70.3% respectively. The culture was continuously perfored for 20 days without any apparent loss in citric acid productivity. Conversely, under the same conditions with a batch shake-flask culture, the maximum productivity, citric acid concentration, and yield were only 63.3 mg L⁻¹ h⁻¹, 4.7 g/L and 51.4%, respectively. Therefore, the results suggest that the bioreactor used in this study could be potentially used for continuous citric acid production from dairy wastewater by applying calcium-alginate immobilizedAspergillus niger.     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

High-frequency plant regeneration by <Emphasis Type="Italic">in vitro</Emphasis> shoot proliferation in cotyledonary node explants of grasspea (<Emphasis Type="Italic">Lathyrus sativus</Emphasis> L.)

Summary Multiple shoots were induced from cotyledonary nodes of grasspea (Lathyrus sativus L.) derived from 7-d-old in vitro seedlings on Murashige and Skoog (MS) medium containing N⁶-benzyladenine (BA), kinetin, or thidiazuron, BA being the most effective. Among the five genotypes tested, shoot proliferation frequency was the highest (93.3%) for IC-120487, giving the maximum number of shoots (11.3 shoots per explant) on MS medium augmented with 2.0 mgl⁻¹ (8.87 μM) BA. Shoot cultures were established by repeatedly subculturing the original cotyledonary nodes on fresh medium after each harvest of the newly formed shoots. Thus 30–40 shoots were obtained in 2 mo. from a single cotyledonary node. Up to 81.8% of the shoots developed roots following transfer to half-strength MS medium containing 0.5 mgl⁻¹ (2.85 μM) indole-3-acetic acid. Plantlets were successfully acclimatized and established in soil.     

Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.