Production of plants resistant to <Emphasis Type="Italic">Alternaria carthami</Emphasis> via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamus tinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 10⁵ spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min⁻¹ mg⁻¹ protein) from organogenesis and 47% (0.23 μmol min⁻¹ mg⁻¹ protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg⁻¹ protein) and 19.5% (145 U mg⁻¹ protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.     

Metabolic fingerprinting for bio-indication of nitrogen responses in <Emphasis Type="Italic">Calluna vulgaris</Emphasis> heath communities

Increased atmospheric deposition of nitrogen (N) over the last 50 years is known to have led to deleterious effects on the health of Calluna vulgaris heathland, with increased proliferation of grasses and loss of species diversity. However, currently it is difficult to attribute damage specifically to N deposition rather than other drivers of change such as inappropriate management. Metabolic fingerprinting using FT-IR offers a rapid, cost-effective and “holistic” means for quantifying foliar biochemistry responses specifically to N deposition. To test the potential of this approach we used a long term lowland heath N addition study in Chesire, England. FT-IR spectra of treated C. vulgaris shoot material showed that responses were detectable above 20 kg N ha⁻¹ year⁻¹. Differentiation was also evident in C. vulgaris metabolic fingerprints due to additional watering. We have shown that FT-IR is able to identify biochemical variations in C. vulgaris related to increases in received N and water. This technique therefore provides a sensitive measure of biochemical change in response to N addition, and allows development towards predictive modelling of N deposition at the landscape level.     

Proteins and Peptidases from Conidia and Mycelia of <Emphasis Type="Italic">Scedosporium apiospermum</Emphasis> Strain HLPB

The conidia–mycelia transformation is an essential step during the life cycle of the fungal human pathogens of the Pseudallescheria boydii complex. In the present study, we have analyzed the protein and peptidase profiles in two distinct morphological stages, conidia and mycelia, of Scedosporium apiospermum sensu stricto. Proteins synthesized by the mycelia, migrating at the ranges of 62–48 and 22–18 kDa, were not detected from the conidial extract. Conidia produced a single cellular peptidase of 28 kDa able to digest copolymerized albumin, while mycelia yielded 6 distinct peptidases ranging from 90 to 28 kDa. All proteolytic enzymes were active at acidic pH and fully inhibited by 1,10-phenanthroline, characterizing these activities as metallo-type peptidases. Quantitative peptidase assay, using soluble albumin, showed a high metallopeptidase production in mycelial cells in comparison with conidia. The regulated expression of proteins and peptidases in different morphological stages of S. apiospermum represents a potential target for isolation of stage-specific markers for biochemical and immunological analysis. Martha Machado Pereira and Bianca Alcantara Silva contributed equally to this work.     

Production of ascorbic acid glucoside by alginate-entrapped mycelia of <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l⁻¹ maltose, 66 g l⁻¹ yeast extract, and 5 g l⁻¹ K₂HPO₄ at 30°C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture, when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l⁻¹ ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l⁻¹ h⁻¹ and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l⁻¹, and the productivity was 1.5 g l⁻¹ h⁻¹. The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately 17 g l⁻¹ of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l⁻¹ h⁻¹ was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography with a final yield of 75%.     

Liquid seaweed extracts identified using <Superscript>1</Superscript>H NMR profiles

Aqueous extracts of Ascophyllum nodosum and several other brown seaweeds are manufactured commercially and widely distributed for use on agricultural crops. The increasingly regulated international trade in such products requires that they be standardized and defined to a degree not previously required. We examined commercially available extracts using quantitative ¹H NMR and principal components analysis (PCA) techniques. Extracts manufactured over a 4-year period using the same process exhibited characteristic profiles that, on PCA, clustered as a discrete group distinct from the other commercial products examined. In addition to recognizing extracts made from different seaweeds, analysis of the ¹H spectra in the 0.35–4.70 ppm region allowed us to distinguish amongst extracts produced from the same algal species by different manufacturers. This result established that the process used to make an extract is an important variable in defining its composition. A comparison of the ¹H NMR integrals for the regions 1.0–3.0 ppm and 3.0–4.38 ppm revealed small but significant changes in the A. nodosum spectra that we attribute to seasonal variation in gross composition of the harvested seaweed. Such changes are reflected in the PCA scores plots and contribute to the scatter observed within the data point cluster observed for Acadian soluble extracts when all data are pooled. Quantitative analysis using ¹H NMR (qNMR) with a certified external standard (caffeine) showed a linear relationship with extract concentration over at least an order of magnitude (2.5–33 mg/mL; R ² > 0.97) for both spectral regions integrated. We conclude that qNMR can be used to profile (or “fingerprint”) commercial seaweed extracts and to quantify the amount of extract present relative to a suitably chosen standard. Issued as NRCC no. 42,652.     

Starch processing wastewater as a new medium for production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Production of Bacillus thuringiensis (Bt) based bioinsecticide was studied by using starch processing wastewater (SPW) as a raw material. Results indicated that the nutrients contained in SPW were sufficient for growth, sporulation and δ-endotoxin production of Bacillus thuringiensis subsp. kurstaki (Btk). The final cell counts and spore counts achieved in SPW medium were 72% and 107% respectively higher than those in the soybean meal based commercial medium. Higher δ-endotoxin yield of 2.67 mg mL⁻¹ and higher entomotoxicity of 1,050 IU μL⁻¹ were also obtained in SPW medium as compared with the commercial medium at the end of fermentation. The morphological observations also revealed that the fermentation cycle of Btk could be shortened in this new medium. This process provides solutions for safe SPW disposal and production of high potency and low cost bioinsecticide.     

Rapid selection of mutants of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> for carbohydrate and fatty acid metabolism by fourier transform infrared spectroscopy and gas chromatography combined with multivariate analysis

Transgene-tagged mutants of Chlamydomonas reinhardtii were generated by random insertional mutagenesis for screening of mutants of carbohydrate and fatty acid metabolism. Approximately 2,500 insertion mutants tagged with the aph7″ gene were produced from one mutagenesis in three weeks. To establish a rapid screening system for numerous insertional lines, whole cell extracts of 100 insertional lines were subjected to Fourier transform infrared spectroscopy (FT-IR) and gas chromatography (GC) analysis combined with multivariate analysis. Mutant lines 28, 67, and 90 showed dramatic differences in the carbohydrate (1,000∼1,200 cm⁻¹) and amide (1,500∼1,700 cm⁻¹) regions of the FT-IR spectrum compared to wild type strain CC-124. Separate GC analysis also showed that 16:0 iso, palmitic acid (16:0), and oleic acid (18:1) were the major fatty acids in the wild type strain. In mutant 80, the relative content ratio of 16:0 iso in total fatty acids was significantly lower than in wild type, whereas the ratios of palmitic acid and oleic acid to 16:0 iso were higher. In mutant 95, the ratio of 16:0 iso to total fatty acids was increased, whereas ratios of palmitic acid and oleic acid to 16:0 iso were decreased. In particular, mutant 57 showed remarkably different fatty acid patterns with novel peaks of long-chain fatty acids having more than 20 carbon atoms. The results of this study show that FT-IR and GC combined with multivariate analysis enable rapid selection of mutants of carbohydrate and fatty acid metabolism in C. reinhardtii.     

Secondary structure of food proteins by Fourier transform spectroscopy in the mid-infrared region

Fourier transform spectroscopy in the mid-infrared (400–5,000 cm⁻¹) (FT-IR) is being recognized as a powerful tool for analyzing chemical composition of food, with special concern to molecular architecture of food proteins. Unlike other spectroscopic techniques, it provides high-quality spectra with very small amount of protein, in various environments irrespective of the molecular mass. The fraction of peptide bonds in α-helical, β-pleated sheet, turns and aperiodic conformations can be accurately estimated by analysis of the amide I band (1,600–1,700 cm⁻¹) in the mid-IR region. In addition, FT-IR measurement of secondary structure highlights the mechanism of protein aggregation and stability, making this technique of strategic importance in the food proteomic field. Examples of applications of FT-IR spectroscopy in the study of structural features of food proteins critical of nutritional and technological performance are discussed.     

Comparison of conditions for mycelial growth of <Emphasis Type="Italic">Lepista sordida</Emphasis> causing fairy rings on <Emphasis Type="Italic">Zoysia matrella</Emphasis> turf to those on <Emphasis Type="Italic">Agrostis palustris</Emphasis> turf

Symptoms of fairy rings caused by Lepista sordida have been reported on Zoysiagrass (Zoysia spp.) turf maintained at fairway height (2 cm), but not on bentgrass (Agrostis spp.) maintained at putting green height (0.5 cm). The mycelia of this fungus inhabit primarily the upper 0–2 cm layer of the soil extending into the thatch. To compare conditions for the mycelial growth in Z. matrella turf to those in A. palustris turf, we examined the effects of nutrients, temperature, water potential, and pH in the field as well as in the laboratory. Greater growth of the mycelia was observed in medium that included hot water extracts from soil of the 0–1 cm zone in Z. matrella turf compared to that from A. palustris. The upper soil layer in Z. matrella turf contained more organic matter from clippings than that in A. palustris. The temperature and water potential of the 0–2 cm soil zone in Z. matrella turf were also more favorable for the mycelial growth. The soil pH values of this zone in Z. matrella turf were less favorable compared to A. palustris but within the range for accelerating mycelial growth. Part of this study was presented orally at the 46th meeting of the Mycological Society of Japan in 2002     

Physico-chemical characterization of a new heteropolysaccharide produced by a native isolate of heterofermentative <Emphasis Type="Italic">Lactobacillus</Emphasis> sp. CFR-2182

A heterofermentative Lactobacillus sp. CFR-2182 was isolated from dahi samples and it was found to produce 8.0 and 20.5 g/L heteropolysaccharide (HePS) in EPS medium (a simplified synthetic medium) and modified MRS broth, respectively, after 72 h at 30°C. The total carbohydrate, reducing sugar and moisture contents of the purified HePS were 74, 10.6 and 2 g, respectively, per 100 g on dry weight basis. The HePS produced in EPS medium had glucose and mannose in 17:1 ratio. The HePS was non-gelling and non-film forming type. It was completely soluble in water and 1 N sodium hydroxide solution. Gel permeation chromatography and HPLC analysis indicated considerable heterogeneity of the HePS, having three fractions with molecular weights ranging from 3.3 × 10⁴ to 1.32 × 10⁶ Da. The enzymatic hydrolysis of the HePS with pullulanase and α-amylase [with α(1→4) linkage] indicated the presence of α(1→6) and traces of α(1→4) linkages, respectively. NMR analysis of the EPS revealed unique chemical shifts.     

Genetic transformation of the medicinal plant <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated method

A high-frequency and simple procedure for Agrobacterium tumefaciens-mediated genetic transformation of the medicinal plant Salvia miltiorrhiza was developed. Leaf discs were pre-cultured on MS medium supplemented with 6.6 μmol l⁻¹ BAP and 0.5 μmol l⁻¹ NAA for one day, then co-cultured with A. tumefaciens strain EHA105 harboring the plasmid pCAMBIA 2301 for three days on the same medium. Regenerated buds were obtained on selection medium (co-culture medium supplemented with 60 mg l⁻¹ kanamycin and 200 mg l⁻¹ cefotaxime) after two cycles’ culture of 10 days each and then transferred to fresh MS medium with 60 mg l⁻¹ kanamycin for rooting. Fifteen days later, the rooted plantlets were obtained and then successfully transplanted to soil. The transgenic nature of the regenerated plants was confirmed by PCR, Southern hybridization analysis and GUS histochemical assay. Averagely, 1.1 independent verified transgenics per explant plated were obtained through this protocol. Adopting this procedure, positive transformed plants could be obtained within 2–3 months from mature seeds germination to transplant to soil, and more than 1,000 transgenic plants with several engineered constructs encoding different genes of interest were produced in our lab in the past two years.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Characterization of Bordetella pertussis growing as biofilm by chemical analysis and FT-IR spectroscopy

Although Bordetella pertussis, the etiologic agent of whooping cough, adheres and grows on the ciliated epithelium of the respiratory tract, it has been extensively studied only in liquid cultures. In this work, the phenotypic expression of B. pertussis in biofilm growth is described as a first approximation of events that may occur in the colonization of the host. The biofilm developed on polypropylene beads was monitored by chemical methods and Fourier transform infrared (FT-IR) spectroscopy. Analysis of cell envelopes revealed minimal differences in outer membrane protein (OMP) pattern and no variation of lipopolysaccharide (LPS) expression in biofilm compared with planktonically grown cells. Sessile cells exhibited a 2.4- to 3.0-fold higher carbohydrate/protein ratio compared with different types of planktonic cells. A 1.8-fold increased polysaccharide content with significantly increased hydrophilic characteristics was observed. FT-IR spectra of the biofilm cells showed higher intensity in the absorption bands assigned to polysaccharides (1,200–900 cm⁻¹ region) and vibrational modes of carboxylate groups (1,627, 1,405, and 1,373 cm⁻¹) compared with the spectra of planktonic cells. In the biofilm matrix, uronic-acid-containing polysaccharides, proteins, and LPS were detected. The production of extracellular carbohydrates during biofilm growth was not associated with changes in the specific growth rate, growth phase, or oxygen limitation. It could represent an additional virulence factor that may help B. pertussis to evade host defenses.     

The complete mitochondrial genome structure of snow leopard <Emphasis Type="Italic">Panthera uncia</Emphasis>

The complete mitochondrial genome (mtDNA) of snow leopard Panthera uncia was obtained by using the polymerase chain reaction (PCR) technique based on the PCR fragments of 30 primers we designed. The entire mtDNA sequence was 16 773 base pairs (bp) in length, and the base composition was: A—5,357 bp (31.9%); C—4,444 bp (26.5%); G—2,428 bp (14.5%); T—4,544 bp (27.1%). The structural characteristics [0] of the P. uncia mitochondrial genome were highly similar to these of Felis catus, Acinonyx jubatus, Neofelis nebulosa and other mammals. However, we found several distinctive features of the mitochondrial genome of Panthera unica. First, the termination codon of COIII was TAA, which differed from those of F. catus, A. jubatus and N. nebulosa. Second, tRNA^{Ser (AGY)}, which lacked the ‘‘DHU’’ arm, could not be folded into the typical cloverleaf-shaped structure. Third, in the control region, a long repetitive sequence in RS-2 (32 bp) region was found with 2 repeats while one short repetitive segment (9 bp) was found with 15 repeats in the RS-3 region. We performed phylogenetic analysis based on a 3 816 bp concatenated sequence of 12S rRNA, 16S rRNA, ND2, ND4, ND5, Cyt b and ATP8 for P. uncia and other related species, the result indicated that P. uncia and P. leo were the sister species, which was different from the previous findings.     

<Emphasis Type="Italic">Hemidesmus indicus</Emphasis> Protects against ethanol-induced liver toxicity

Alcoholic liver disease (ALD) is one of the most common diseases in modern society. A large number of studies are in progress aiming to identify natural substances that would be effective in reducing the severity of ALD. Although there are currently a number of drugs on the market, their long-term use can have numerous side effects. Hemidesmus indicus is an indigenous Ayurvedic medicinal plant used in soft drinks in India. In this study, we examined the effects of its ethanolic root extract on experimental liver damage in order to evaluate its hepatoprotective effects against hepatotoxicity induced in rats by ethanol at a dosage of 5 g/kg body weight for 60 days. The H. indicus root extract was given at a dose of 500 mg/kg body weight for the last 30 days of the experiment. The animals were monitored for food intake and weight gain. The liver was analysed for the degree of lipid peroxidation using thiobarbituric acid reactive substances (TBARS) and antioxidant status using the activities of glutathione-depedendant enzymes. The degree of liver damage was analysed using serum marker enzyme activities, the total protein, albumin, globulin, ceruloplasmin and liver glycogen contents, and the A/G ratio. The Fourier transform infrared spectra (FT-IR) of the liver tissues were recorded in the region of 4000–400 cm⁻¹. The ethanol-fed rats showed significantly elevated liver marker enzyme activities, lipid peroxidation levels and reduced antioxidant levels as compared to the control rats. Oral administration of H. indicus for the latter 30 days resulted in an increased food intake and weight gain, decreased TBARS levels, near normal levels of glutathione-dependent enzymes, increased total protein, albumin, globulin and liver glycogen contents, an increased A/G ratio, and decreased liver marker enzyme activities and ceruloplasmin levels. The relative intensity of the liver FT-IR bands for the experimental groups were found to be altered significantly (p < 0.05) compared to the control samples. For the group that had H. indicus co-administered with ethanol, the intensity of the bands was near normal. Moreover, the results of the FT-IR study correlated with our biochemical results.     

Enhanced iturin A production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and its effect on suppression of the plant pathogen <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The behavior of Streptomyces peucetius var. caesius N47 was studied in a glucose limited chemostat with a complex cultivation medium. The steady-state study yielded the characteristic constants μ _max over 0.10 h⁻¹, Y _XS 0.536 g g⁻¹, and m_S 0.54 mg g⁻¹ h⁻¹. The product of secondary metabolism, ɛ-rhodomycinone, was produced with characteristics Y _PX 12.99 mg g⁻¹ and m _P 1.20 mg g⁻¹ h⁻¹. Significant correlations were found for phosphate and glucose consumption with biomass and ɛ-rhodomycinone production. Metabolic flux analysis was conducted to estimate intracellular fluxes at different dilution rates. TCA, PPP, and shikimate pathway fluxes exhibited bigger values with production than with growth. Environmental perturbation experiments with temperature, airflow, and pH changes on a steady-state chemostat implied that an elevation of pH could be the most effective way to shift the cells from growing to producing, as the pH change induced the biggest transient increase to the calculated ɛ-rhodomycinone flux.     

Secondary structure estimation of recombinant <Emphasis Type="Italic">psbH</Emphasis>, encoding a photosynthetic membrane protein of cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The PsbH protein of cyanobacterium Synechocystis sp. PCC 6803 was expressed as a fusion protein with glutathione-S transferase (GST) in E. coli grown on a mineral medium enriched in ¹⁵N isotope. After enzymatic cleavage of the fusion protein, the ¹H-¹⁵N-HSQC spectrum of PsbH protein in presence of the detergent β-D-octyl-glucopyranoside (OG) was recorded on a Bruker DRX 500 MHz NMR spectrometer equipped with a 5 mm TXI cryoprobe to enhance the sensitivity and resolution. Non-labelled protein was used for secondary structure estimation by deconvolution from circular dichroism (CD) spectra. Experimental results were compared with our results from a structural model of PsbH using a restraint-based comparative modelling approach combined with molecular dynamics and energetic modelling. We found that PsbH shows 34–38% α-helical structure (Thr36-Ser60), a maximum of around 15% of β-sheet, and 12–19% of β-turn.     

Quantitative conformational analysis of cytochromec bound to phospholipid vesicles studied by resonance Raman spectroscopy

Resonance Raman spectra have been recorded from ferri-cytochromec bound to phospholipid vesicles composed of dimyristoyl phosphatidylglycerol (DMPG), dioleoyl phosphatidylglycerol (DOPG) or dioleoyl phosphatidylglycerol-dioleoyl phasphatidylcholine (DOPG-OPC) (70 : 30 mole/mole). Lipid binding induces very significant conformational changes in the protein molecule. The resonance Raman spectra differ in their content of bands originating from two different conformational species, I and II, of the protein, and from two different spin and coordination states of the heme in conformation II. Data of sufficiently high precision were obtained that the spectra of the individual species could be quantitated by a constraint interative fitting routine using single Lorentzian profiles. In the high frequency, or marker band region (1200 to 1700 cm⁻¹), the frequencies, half widths and relative intensities of the individual bands could be estimated from previous surface enhanced resonance Raman measurements on cytochromec adsorbed on a silver electrode. These were then further optimized to yield both the spectral parameters and relative contents of the different species. In the low frequency, or finger-print, region (200 to 800 cm⁻¹), the spectral parameters of the individual species were obtained from difference spectra derived by sequential subtraction between the spectra of ferri-cytochromec in the three different lipid systems, using the relative proportions of the species derived from the marker band region. These parameters were then subsequently refined by iterative optimization. The optimized spectral parameters in both frequency regions for the six-coordinated low spin states I and II, and for the five-coordinated high spin state II are presented. The proportion of state II, in which hence the heme crevice assumes an open structure, and of the five-coordinated high spin configuration, is found to increase on binding ferri cytochromec to negatively charged lipid vesicles. The extent of this conformational change increases in the order: DOPG-DOPC<DOPG<DMPG, with a parallel decrease of the proportion of the conformational state I, whose structure is similar to that of the uncomplexed ferri-cytochrome c in solution. Similar conformational changes are found for ferro-cytochromec compared to those obtained with the oxidized species on binding to lipids. The present work is essential for studies which seek to analyze, in any detailed fashion, the conformational transitions in the heme protein which take place in response to changes in the lipid environment.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated genetic transformation in tea (<Emphasis Type="Italic">Camellia sinensis</Emphasis> [L.] O. Kuntze)

We have developed a system to produce transgenic plants in tea (Camelia sinensis [L.] O. Kuntze) viaAgrobacterium tumefaciens-mediated transformation of embryogenic calli. Cotyledon-derived embryogenic callus cultures were cocultivated with anA. tumefaciens strain (AGL 1) harboring a binary vector carrying the hygromycin phosphotransferase (hpt II), glucuronidase (uid A), and green fluorescent protein (GFP) genes in the tDNA region. Following cocultivation, embryogenic calli were cultured in medium containing 500 mg/L carbenicillin for 1 wk and cultured on an antibiotic selection medium containing 75 mg/L hygromycin for 8–10 wk. Hygromycin-resistant somatic embryos were selected. The highest production efficiency of hygromycin-resistant calli occurred with cocultivation for 6–7 d in the presence of 400 μM acetosyringone (AS). Hygromycin-resistant somatic embryos developed into complete plantlets in regeneration medium containing half-strength Murashige and Skoog (MS) salts with 1 mg/L benzyl amino purine (BAP) and 9 mg/L giberellic acid (GA₃). Transformants were subjected to GFP expression analysis, β-glucuronidase (GUS) histochemical assay, PCR analysis, and Southern hybridization to confirm gene integration.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of embryogenic tissues of hybrid firs (<Emphasis Type="Italic">Abies</Emphasis> spp.) and regeneration of transgenic emblings

A genetic transformation system has been developed for selected embryogenic cell lines of hybrids Abies alba × A. cephalonica (cell lines AC2, AC78) and Abies alba × A. numidica (cell line AN72) using Agrobacterium tumefaciens. The cell lines were derived from immature or mature zygotic embryos on DCR medium containing BA (1 mg l⁻¹). The T-DNA of plant transformation vector contained the β-glucuronidase reporter gene under the control of double dCaMV 35S promoter and the neomycin phosphotransferase selection marker gene driven by the nos promoter. The regeneration of putative transformed tissues started approximately 1 week after transfer to the selection medium containing 10 mg geneticin l⁻¹. GUS activity was detected in most of the geneticin-resistant sub-lines AN72, AC2 and AC78, and the transgenic nature of embryogenic cell lines was confirmed by PCR approach. Plantlet regeneration from PCR-positive embryogenic tissues has been obtained as well. The presence of both gus and nptII genes was confirmed in 11 out of 36 analysed emblings.