Efficient separation and purification of allophycocyanin from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

An investigation of <Emphasis Type="Italic">Clostridium</Emphasis> species present in nutraceutical preparations of <Emphasis Type="Italic">Arthrospira platensis</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) for human consumption

The presence of the anaerobic spore former Clostridium in Arthrospira platensis destined for human consumption is generally not assessed during quality assurance procedures. As this nutraceutical is administered as complementary medicine to the immunocompromised, this study aimed to investigate the presence of these potential pathogens. Anaerobic counts performed on tablets from a single manufacturer indicated an excess of 10⁵ CFU/endospores g⁻¹ tablet for three different A. platensis batches. Tests for coliforms for use as “indicators” of pathogens in the tablets were negative. Using classic culture techniques, five species of Clostridium were isolated. Subsequent use of PCR-denaturing gradient gel electrophoresis (DGGE) fingerprinting of tablets showed a divergent microbial population, with a predominance of anaerobic endospore formers, including Clostridium. Sequencing of a 1.5 kb 16S rDNA clone library and phylogenetic analyses of prominent operational taxonomic units confirmed the presence of an additional five Clostridium spp. and other genera in the tablets. A composite molecular ladder, using 16S rRNA DGGE amplicons of 17 representative bacterial species was constructed to assist in identifying anaerobes present in tablets sourced from three different A. platensis manufacturers. Results indicated that commercial A. platensis preparations were contaminated with potentially hazardous clostridia and other anaerobic species. Results suggest that certain commercial A. platensis preparations require stringent microbial quality assurance measures to ensure safe use as a nutraceutical for the immunocompromised and the general public.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Benzyladenine Promotes Flowering in <Emphasis Type="Italic">Doritaenopsis</Emphasis> and <Emphasis Type="Italic">Phalaenopsis</Emphasis> Orchids

Two experiments were performed to determine how application of the cytokinin benzyladenine (BA) influenced flowering in Doritaenopsis and Phalaenopsis orchid clones. In the first experiment, two vegetative orchid clones growing in 15-cm pots were transferred from a 28°C greenhouse that inhibited flowering to a 23°C greenhouse for flower induction (day 0). A foliar spray (0.2 L m⁻²) containing BA at 100, 200, or 400 mg L⁻¹ or 25, 50, or 100 mg L⁻¹ each of BA and gibberellins A₄ + A₇ (BA+GA) was applied on days 0, 7, and 14. Plants treated with BA alone at 200 or 400 mg L⁻¹ had a visible inflorescence 3–9 days earlier and had a mean of 0.7–3.5 more inflorescences and 3–8 more flowers per plant than nontreated plants. The application of BA+GA had no effect on inflorescence number and total flower number at the rates tested. In the second experiment, three orchid clones received a single foliar spray of BA at 200 mg L⁻¹ at six time points relative to time of transfer from 29°C to 23°C (−1, 0, +1, +2, +4, or +6 weeks). A separate group of plants received a BA application at week 0 but was maintained at 29°C. Inflorescence number was greatest in all three orchid clones when plants were treated with BA 1 week after the temperature transfer. Plants that were sprayed with BA and maintained at 29°C did not initiate inflorescences. The promotion of flowering by the application of BA suggests that cytokinins at least partially regulate inflorescence initiation of Doritaenopsis and Phalaenopsis, but its promotion is conditional and BA application cannot completely substitute for an inductive low temperature.     

Insecticide-tolerant and plant growth promoting <Emphasis Type="Italic">Bradyrhizobium</Emphasis> sp. (<Emphasis Type="Italic">vigna</Emphasis>) improves the growth and yield of greengram [<Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek] in insecticide-stressed soils

This study was designed to identify rhizobial strains specific to greengram expressing higher tolerance against insecticides, fipronil and pyriproxyfen, and synthesizing plant growth regulators even amid insecticide-stress. Of the 50 bradyrhizobial isolates, the Bradyrhizobium sp. strain MRM6 showed tolerance up to 1,600 μg mL⁻¹ against each of fipronil and pyriproxyfen. The tolerant Bradyrhizobium sp. (vigna) produced plant growth promoting substances in substantial amounts, both in the presence and absence of insecticides. The strain MRM6 was further used to investigate its impact on greengram grown in soils treated with 200 (the recommended dose), 400 and 600 μg kg⁻¹ soil of fipronil and 1,300 (the recommended dose), 2,600 and 3,900 μg kg⁻¹ soil of pyriproxyfen. Fipronil at 600 μg kg⁻¹ soils and pyriproxyfen at 3,900 μg kg⁻¹ soils had greatest toxic effects and decreased plant biomass, symbiotic efficiency, nutrient uptake and seed yield of greengram plants. The Bradyrhizobium sp. (vigna) inoculant when used with fipronil and pyriproxyfen significantly increased the measured parameters compared to the plants grown in soils treated solely with the same concentration of each insecticide. This study inferred that the Bradyrhizobium sp. (vigna) strain MRM6 may be exploited as bio-inoculant to increase the productivity of greengram exposed to insecticide-stressed soils.     

Tissue culture of <Emphasis Type="Italic">Sinningia speciosa</Emphasis> and analysis of the <Emphasis Type="Italic">in vitro</Emphasis>-generated <Emphasis Type="Italic">tricussate whorled phyllotaxis</Emphasis> (<Emphasis Type="Italic">twp</Emphasis>) variant

Two protocols were developed for the efficient regeneration of Sinningia speciosa from leaf explants via two developmental pathways. The first method involved formation of callus and buds, followed by subsequent root growth, in Murashige and Skoog medium (MS) containing 2.0 mg l⁻¹ 6-benzylaminopurine (BA) and 0.2 mg l⁻¹ α-naphthalene acetic acid (NAA), with a regeneration efficiency of 99.0%. The second method involved producing callus and roots, followed by subsequent formation of buds, in MS medium supplemented with 1.0–5.0 mg l⁻¹ NAA, and resulted in a regeneration efficiency of 90.4%. Our experiments indicate that the root-first pathway resulted in a lower plant regeneration efficiency. Through five continual generations using the buds-first method, a total of 215 regenerated plants were obtained in the last generation, and eight exhibited a phenotype we named tricussate whorled phyllotaxis (twp). Six of the regenerated twp variant plants maintained their tricussate whorled phyllotaxis phenotype, showing no other abnormalities, while one reverted to a wild type before flowering and another formed two rounds of sepals. Physiological analysis revealed that the twp plants responded differently than wild type to exogenous NAA and 2,3,5-triiodobenzoic acid (TIBA), while high-performance liquid chromatography (HPLC) analysis showed that the levels of endogenous indole-3-acetic acid (IAA) and gibberellin (GA) were lower in twp than wild-type plants. These results suggest that the formation of the twp mutant may be related to phytohormones and that the twp variant could be an important material for investigating the molecular mechanism of plant phyllotaxis patterning.     

Detrimental effect of increasing sugar concentrations on ethanol production from maize or decorticated sorghum mashes fermented with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> or <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

The efficiency of ethanol fermentation, as affected by grain source (maize and decorticated red sorghum), total sugar concentration (13 or 20° Plato) and type of microorganism (Saccharomyces cerevisiae or Zymomonas mobilis) was studied. Maize mashes yielded 0.32 l ethanol kg⁻¹ ground grain whereas mashes prepared with decorticated red sorghum produced 0.28 l ethanol kg⁻¹. Both microorganisms yielded similar amounts of ethanol. However, high-gravity mashes (20° Plato) yielded lower amounts of ethanol compared to counterparts adjusted to 13° Plato (0.28 vs. 0.22 l ethanol kg⁻¹ ground grains). In decorticated sorghum mashes adjusted to 20° P, Z. mobilis produced 40 ml kg⁻¹ more ethanol compared to S. cerevisiae. In addition, Z. mobilis had a lower dependency on nitrogenous compounds.     

Control of <Emphasis Type="Italic">Bemisia tabaci</Emphasis> and <Emphasis Type="Italic">Frankliniella occidentalis</Emphasis> in cucumber by <Emphasis Type="Italic">Amblyseius swirskii</Emphasis>

Bemisia tabaci Genn. (Hemiptera: Aleyrodidae) and Frankliniella occidentalis (Thysanoptera: Thripidae) are major pests in greenhouse grown cucumber crops. Recently, Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae) was shown an effective biological control agent of both pests. Hence, perhaps both pests can be controlled simultaneously by this predator. However, with simultaneous infestation of both pests, synergistic effects, or interference could affect biological control and perhaps require changes in release rates of the predator. Thus, the aim of the present study was to evaluate different release rates of A. swirskii to control both pests under a worst case scenario of rapid immigration into a cucumber greenhouse. Two experiments were conducted, one simulating the influx of whiteflies alone (whitefly experiment) and the other immigration of whiteflies and thrips together (whitefly plus thrips experiment). Three treatments were compared in the whitefly experiment: (1) B. tabaci alone, (2) B. tabaci + 25 A. swirskii m⁻² and (3) B. tabaci + 75 A. swirskii m⁻². The high release rate was more effective than the low rate in controlling B. tabaci alone. The high rate was subsequently tested against B. tabaci and F. occidentalis for the whitefly and thrips experiment in which five treatments were compared: (1) B. tabaci alone, (2) F. occidentalis alone, (3) B. tabaci + 75 A. swirskii m⁻², (4) F. occidentalis + 75 A. swirskii m⁻² and (5) B. tabaci + F. occidentalis + 75 A. swirskii m⁻². This rate of A. swirskii controlled whiteflies and thrips either alone or together. Therefore, 75 A. swirskii m⁻² should be an adequate rate for controlling both pests either alone or simultaneously in cucumber greenhouses.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

<Emphasis Type="SmallCaps">l</Emphasis>-Proline nutrition and catabolism in <Emphasis Type="Italic">Staphylococcus saprophyticus</Emphasis>

Staphylococcus saprophyticus strains ATCC 15305, ATCC 35552, and ATCC 49907 were found to require l-proline but not l-arginine for growth in a defined culture medium. All three strains could utilize l-ornithine as a proline source and contained l-ornithine aminotransferase and Δ¹-pyrroline-5-carboxylate reductase activities; strains ATCC 35552 and ATCC 49907 could use l-arginine as a proline source and had l-arginase activity. The proline requirement also could be met by l-prolinamide, l-proline methyl ester, and the dipeptides l-alanyl-l-proline and l-leucyl-l-proline. The bacteria exhibited l-proline degradative activity as measured by the formation of Δ¹-pyrroline-5-carboxylate. The specific activity of proline degradation was not affected by addition of l-proline or NaCl but was highest in strain ATCC 49907 after growth in Mueller–Hinton broth. A membrane fraction from this strain had l-proline dehydrogenase activity as detected both by reaction of Δ¹-pyrroline-5-carboxylate with 2-aminobenzaldehyde (0.79 nmol min⁻¹ mg⁻¹) and by the proline-dependent reduction of p-iodonitrotetrazolium (20.1 nmol min⁻¹ mg⁻¹). A soluble fraction from this strain had Δ¹-pyrroline-5-carboxylate dehydrogenase activity (88.8 nmol min⁻¹ mg⁻¹) as determined by the NAD⁺-dependent oxidation of dl-Δ¹-pyrroline-5-carboxylate. Addition of l-proline to several culture media did not increase the growth rate or final yield of bacteria but did stimulate growth during osmotic stress. When grown with l-ornithine as the proline source, S. saprophyticus was most susceptible to the proline analogues L-azetidine-2-carboylate, 3,4-dehydro-dl-proline, dl-thiazolidine-2-carboxylate, and l-thiazolidine-4-carboxylate. These results indicate that proline uptake and metabolism may be a potential target of antimicrobial therapy for this organism.     

Associations between common variants in <Emphasis Type="Italic">GC</Emphasis> and <Emphasis Type="Italic">DHCR7</Emphasis>/<Emphasis Type="Italic">NADSYN1</Emphasis> and vitamin D concentration in Chinese Hans

Recent studies have identified common variants in or near GC, CYP2R1 and NADSYN1/DHCR7 to be associated with 25-hydroxyvitamin D [25(OH)D] levels in European populations. We aimed to examine whether these variants also influence 25(OH)D levels in Chinese. Seven common variants were successfully genotyped and tested for associations with plasma 25(OH)D levels in a population-based cohort of 3,210 Chinese Hans from Beijing and Shanghai. Six common variants at GC (rs4588, rs7041, rs2282679 and rs1155563) and NADSYN1/DHCR7 (rs3829251 and rs1790349) loci were all significantly associated with lower plasma 25(OH)D levels (−0.036 ≤ β ≤ −0.076 per risk-allele, P ≤ 5.7 × 10⁻⁵), while CYP2R1-rs2060793 showed a trend toward association with 25(OH)D levels in the Shanghai subpopulation (P = 0.08), but not in the Beijing subpopulation (P = 0.82). Haplotype-based association analyses of the four GC variants showed that only the haplotype that contained all risk-alleles (TACC) was significantly associated with lower plasma 25(OH)D levels (β = −0.085, P = 2.3 × 10⁻⁹), while the haplotype containing the risk-alleles of rs4588 and rs2282679 (TATC) was marginally associated with lower 25(OH)D levels (β = −0.054, P = 0.0562) when compared with GCTA haplotype carrying the four protective alleles. Most notably, conditional analyses showed that only GC-rs4588 and GC-rs2282679 (r ² = 0.97) remained significantly associated with 25(OH)D concentrations (P ≤ 1.9 × 10⁻⁵) after adjusting for the other two SNPs in GC. In conclusion, GC and NADSYN1/DHCR7 loci individually and collectively contribute to variation in plasma vitamin D levels in Chinese Hans.     

In vitro induction of inflorescence in <Emphasis Type="Italic">Dioscorea</Emphasis><Emphasis Type="Italic">zingiberensis</Emphasis>

Inflorescence induction and morphogenesis of regenerated flowers were investigated in vitro in Dioscorea zingiberensis C. H. Wright. Inflorescence induction was influenced by the type and concentration of phytohormones. When floral bud explants were incubated on a Murashige and Skoog medium containing a combination of 2.0 mg l⁻¹ 6-benzyladenine and 0.5 mg l⁻¹ indole-3-butyric acid, the highest frequency of inflorescence induction was observed. However, in the presence of gibberellic acid, induction efficiency was reduced although node length of inflorescence was increased. Ontogenetic studies revealed that the inflorescence primordia originated directly from axillary epidermal cells of the perianth and bract of the explants after 7 days. In vitro, male flowers developed normally and blossomed after 90–100 days. In addition, some bisexual flowers were observed. These results demonstrated that there were differences in sexual differentiation of floral buds in vitro compared with that in vivo.     

Photoregulation of morphological structure and its physiological relevance in the cyanobacterium <Emphasis Type="Italic">Arthrospira</Emphasis> (<Emphasis Type="Italic">Spirulina</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

The spiral structure of the cyanobacterium Arthrospira (Spirulina) platensis (Nordst.) Gomont was previously found to be altered by solar ultraviolet radiation (UVR, 280–400 nm). However, how photosynthetic active radiation (PAR, 400–700 nm) and UVR interact in regulating this morphological change remains unknown. Here, we show that the spiral structure of A. platensis (D-0083) was compressed under PAR alone at 30°C, but that at 20°C, the spirals compressed only when exposed to PAR with added UVR, and that UVR alone (the PAR was filtered out) did not tighten the spiral structure, although its presence accelerated morphological regulation by PAR. Their helix pitch decreased linearly as the cells received increased PAR doses, and was reversible when they were transferred back to low PAR levels. SDS-PAGE analysis showed that a 52.0 kDa periplasmic protein was more abundant in tighter filaments, which may have been responsible for the spiral compression. This spiral change together with the increased abundance of the protein made the cells more resistant to high PAR as well as UVR, resulting in a higher photochemical yield.     

Tumor-inhibitory and liver-protective effects of <Emphasis Type="Italic">Phellinus igniarius</Emphasis> extracellular polysaccharides

The tumor-inhibitory and liver-protective effects of crude extracellular polysaccharides (EPS) extracted from the liquid mycelial culture of the mushroom Phellinus igniarius were studied in mice. The mice were injected with murine sarcoma S180 and murine hepatoma H22. Crude EPS at 100, 200, 400 mg kg⁻¹ body weight was administered to EPS groups each day in the twelve consecutive days. The result showed that EPS 200 mg kg⁻¹ body weight significantly inhibited S180 and H22 at 65.0 and 46.3%, respectively. Moreover, EPS could not only keep the numbers of WBC, RBC, PLT and the concentration of HGB in a normal range, but also normalize the activities of AST, ALT and ALP. For example, in EPS-treated mice, AST significantly reduced with the percentage of A/G reverse in S180 (P < 0.05) and H22 (P < 0.01) when the mice took EPS 200 mg kg⁻¹ body weight. In conclusion, it was remarkable that P. igniarius EPS exhibited antitumor activity related to dosage and protected liver function by sustaining the blood routine as well as keeping the blood biochemical indexes normal.     

Synergistic effect of green tea extract and probiotics on the pathogenic bacteria, <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>

The aim of this study was to assess the effect of a commercial green tea extract (TEAVIGO™) on the microbial growth of three probiotic strains (Lactobacillus and Bifidobacterium), as well as three pathogenic bacteria. MIC and co-culture studies were performed. The MICs of the green tea extract against Staphylococcus aureus and Streptococcus pyogenes (100 μg ml⁻¹) were considerably lower than those against the probiotic strains tested (>800 μg ml⁻¹) and Escherichia coli (800 μg ml⁻¹). In co-culture studies, a synergistic effect of the probiotic strains and the green tea extract was observed against both Staph. aureus and Strep. pyogenes. Green tea extract in combination with probiotics significantly reduced the viable count of both pathogens at 4 h and by 24 h had completely abolished the recovery of viable Staph. aureus and Strep. pyogenes. These reductions were more significant than the reductions induced by probiotics or green tea extracts used separately. These results demonstrate the potential for combined therapy using the green tea extract plus probiotics on microbial infections caused by Staph. aureus and Strep. pyogenes. As probiotics and the green tea extract are derived from natural products, treatment with these agents may represent important adjuncts to, or alternatives to, conventional antibiotic therapy.     

In vitro regeneration of the important North American oak species <Emphasis Type="Italic">Quercus alba</Emphasis>, <Emphasis Type="Italic">Quercus bicolor</Emphasis> and <Emphasis Type="Italic">Quercus rubra</Emphasis>

North American oak species, with their characteristic strong episodic seasonal shoot growth, are highly problematic for clonal micropropagation, resulting in the inability to achieve a stabilized shoot multiplication stage. The potential for initiating and proliferating shoot cultures derived from Quercus alba, Q. bicolor and Q. rubra explants was investigated, and a micropropagation method for these species was developed. Branch segments from 6 to 7-year-old trees were forced-flushed and the forced shoots were used as source of explants for culture initiation. A consistent shoot multiplication stage was achieved, in 13 of the 15 genotypes established in vitro, although marked differences occurred in explants from different genotypes/species. The control of efficient shoot multiplication involved the culture of decapitated shoots in a stressful horizontal position on cytokinin-containing medium with a sequence of transfers within a 6-week subculture cycle, which was beneficial to overcoming the episodic character of shoot growth. During each subculture cycle, the horizontally placed explants were cultured on media containing 0.2 mg l⁻¹ benzyladenine (BA) for 2 weeks with two successive transfers (2 weeks each) to fresh medium with 0.1 mg l⁻¹ BA, giving a 6-week subculture cycle. The general appearance and vigor of Q. alba and Q. bicolor shoot cultures were improved by the inclusion of both 0.1 mg l⁻¹ BA and 0.5 mg l⁻¹ zeatin in the medium used for the second transfer within the 6-week subculture cycle. Addition of AgNO₃ (3 mg l⁻¹) to the shoot proliferation medium of Q. rubra had a significant positive effect on shoot development pattern by reducing deleterious symptoms, including shoot tip necrosis and early senescence of leaves. The three species showed acceptable in vitro rooting rates by culturing microcuttings in medium containing 25 mg l⁻¹ indolebutyric acid for 48 h with subsequent transfer to auxin-free medium supplemented with 0.4% activated charcoal. Although an initial 5-day dark period generally improved the rooting response, it was detrimental to the quality of regenerated plantlets. However, activated charcoal stimulated not only the rooting frequencies, but it also enhanced plant quality, as evidenced by root, shoot and leaf growth.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Single-step chromatography for simultaneous purification of C-phycocyanin and allophycocyanin with high purity and recovery from <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

The cyanobacterium Spirulina (Arthrospira) platensis is a good source of phycobiliprotein purification. C-phycocyanin (C-PC) is the major phycobiliprotein, while allophycocyanin (APC) is less abundant in S. platensis. Previously reported methods for C-PC purification are only able to offer either high purity or high efficiency. This paper describes one-step anion exchange chromatography method with continuous pH gradient elution for simultaneous purification of C-PC and APC with high purity and high recovery. Crude C-PC and APC were extracted and concentrated by ammonium sulfate fractionation at saturation of 25% and 60%, then purified on a DEAE-Sepharose Fast Flow chromatography column with continuous pH gradient elution from pH 5.0 to 3.6. After this single-step chromatography, C-PC and APC with high purity and recovery were simultaneously obtained. The purity ratios of C-PC and APC reached 5.59 (A₆₂₀/A₂₈₀) and 5.19 (A₆₅₀/A₂₈₀), respectively. Their purity was further demonstrated by electrophoresis and fluorescence emission spectroscopy. Moreover, the total recovery yield of pure C-PC and APC were 67.04% and 80.0%, representing 111.83 and 29.28 mg·g⁻¹ lyophilized weight, respectively. The obtained C-PC and APC remained stable over a pH range of 4–9. This purification method for high purity and recovery of C-PC and APC proved to be fairly efficient compared with previously reported methods.