Stable chloroplast transformation in cabbage (<Emphasis Type="Italic">Brassica oleracea </Emphasis>L. var. <Emphasis Type="Italic">capitata </Emphasis>L.) by particle bombardment

The objectives of this research were first to isolate plastid gene sequences from cabbage (Brassica oleracea L. var. capitata L.), and to establish the chloroplast transformation technology of Brassica. A universal transformation vector (pASCC201) for Brassica chloroplast was constructed with trnV–rrn16S (left) and trnI–trnA–rrn23S (right) of the IR_A region as a recombination site for the transformed gene. In transforming plasmid pASCC201, a chimeric aadA gene was cloned between the rrn16S and rrn23S plastid gene borders. Expression of aadA confers resistance to spectinomycin and streptomycin antibiotics. The uidA gene was also inserted into the pASCC201 and transferred into the leaf cells of cabbage via particle gun mediated transformation. Regenerated plantlets were selected by 200 mg/l spectinomycin and streptomycin. After antibiotic selection, the regeneration percentage of the two cabbage cultivars was about 2.7–3.3%. The results of PCR testing and Southern blot analysis confirmed that the uidA and aadA genes were present in the chloroplast genome via homologously recombined. Northern blot hybridizations, immunoblotting and GUS histochemical assays indicated that the uidA gene were stable integrated into the chloroplast genome. Foreign protein was accumulated at 3.2–5.2% of the total soluble protein in transgenic mature leaves. These results suggest that the expression of a variety of foreign genes in the chloroplast genome will be a powerful tool for use in future studies.     

Production and cytogenetic characterization of intertribal somatic hybrids between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Isatis indigotica</Emphasis> and backcross progenies

Intertribal somatic hybrids between Brassica napus (2n = 38, AACC) and a dye and medicinal plant Isatis indigotica (2n = 14, II) were obtained by fusions of mesophyll protoplasts. From a total of 237 calli, only one symmetric hybrid (S2) and five asymmetric hybrids (As1, As4, As6, As7 and As12) were established in the field. These hybrids showed some morphological variations and had very low pollen fertility. Hybrids S2 and As1 possessed 2n = 52 (AACCII), the sum of the parental chromosomes, and As12 had 2n = 66 (possibly AACCIIII). Hybrids As4, As6 and As7 were mixoploids (2n = 48–62). Genomic in situ hybridization analysis revealed that pollen mother cells at diakinesis of As1 contained 26 bivalents comprising 19 from B. napus and 7 from I. indigotica and mainly showed the segregation 26:26 at anaphase I (AI) with 7 I. indigotica chromosomes in each polar group. Four BC₁ plants from As1 after pollinated by B. napus resembled mainly B. napus in morphology but also exhibited some characteristics from I. indigotica. These plants produced some seeds on selfing or pollination by B. napus. They had 2n = 45 (AACCI) and underwent pairing among the I. indigotica chromosomes and/or between the chromosomes of two parents at diakinesis. All hybrids mainly had the AFLP banding patterns from the addition of two parents plus some alterations. B. napus contributed chloroplast genomes in majority of the hybrids but some also had from I. indigotica. Production of B. napus–I. indigotica additions would be of considerable importance for genome analysis and breeding.     

Expression of the glutathione <Emphasis Type="Italic">S</Emphasis>-transferase gene (NT107) in transgenic <Emphasis Type="Italic">Dianthus superbus</Emphasis>

Using an Agrobacterium-mediated transformation method based on wounding cultured immature seeds with carborundum (600 mesh) in liquid, auxin-regulated tobacco glutathione S -transferase (GST) (NT107) constructs were used to transform Dianthus superbusL. A 663 bp DNA band was found in the transgenic plant genome by PCR analysis using NT107-1 and NT107-2 primers, and a Southern blot analysis showed that the DIG-labelled GST gene was hybridized to the expected amplified genomic DNA fragment from transgenic D. superbus. An overexpression of NT107 led to a twofold increase in GST-specific activity compared to the non-transgenic control plants, and the GST overexpression plants showed an enhanced acclimatization in the soil. To investigate whether an increased expression of GST could affect the resistance of photosynthesis to environmental stress, these plants were subjected to drought and various light intensities from 100 to 3000 mol m^–2s^–1. Copper accumulation and the translocation rate were also analysed in the transgenic lines, and the GST overexpression plants were found to synthesize phytochelatin (PC), which functions by sequestering and detoxifying excess copper ions.These two authors contributed equally to this work     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> bulblet regeneration from immature embryos of endangered <Emphasis Type="Italic">Sternbergia fischeriana</Emphasis>

Sternbergia fischeriana is an endangered geophyte and therefore in vitro micropropagation of this plant will have great importance for germplasm conservation and commercial production. Bulb scale and immature embryo explants of S. fischeriana were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. Immature embryos produced higher number of bulblets than bulb scales. Large numbers of bulblets were regenerated (over 80 bulblets/explants) from immature embryos on Murashige and Skoog (MS) medium supplemented with 4 mg l^–1 6-benzylaminopurine (BA) and 0.25 mg l^–1 -naphthaleneacetic (NAA) or 2 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D) after 14 months of culture initiation. Regenerated bulblets were kept at 5 °C for 5 weeks and then transplanted to a potting mixture.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Electrofusion of protoplasts from <Emphasis Type="Italic">Solanum tuberosum</Emphasis>, <Emphasis Type="Italic">S. bulbocastanum</Emphasis> and <Emphasis Type="Italic">S. pinnatisectum</Emphasis>

This paper discusses a number of experiments performed, involving the fusion by an electric field of mesophyll protoplasts from Solanum tuberosum cv. Bintje, S. tuberosum dihaploid clones 243, 299 and the wild tuberous disease-resistant species S. bulbocastanum and S. pinnatisectum. Three fusion experiments (S. bulbocastanum + S. tuberosum dihaploid 243, S. pinnatisectum + S. tuberosum cv. Bintje and S. pinnatisectum + S. tuberosum dihaploid 299) yielded 542 calli, the 52 ones of which produced shoots. Obtained regenerants were estimated by the flow-cytometry (FC) and RAPD analysis to determine hybrid plants.The utilisation of the FC as a useful method for detecting somatic hybrids is also discussed in this paper. The combination S. bulbocastanum + S. tuberosum dihaploid 243 led to the creation of eight somatic hybrids, the combination S. pinnatisectum + S. tuberosum cv. Bintje yielded four somatic hybrids and the combination S. pinnatisectum + S. tuberosum dihaploid 299 resulted in no hybrid regenerants. Morphology in vitro, growth vigour and production of tuber-like structures were evaluated in hybrid plants. Plants were transferred in vivo for further estimation (acclimatization, habitus evaluation and tuberization ability).     

<Emphasis Type="Italic">SulP</Emphasis>, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Genomic, proteomic, phylogenetic and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the green alga Chlamydomonas reinhardtii are described. This nuclear-encoded sulfate permease gene (SulP) contains four introns, whereas all other known chloroplast sulfate permease genes lack introns and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contains seven transmembrane domains and two large hydrophilic loops. This novel prokaryotic-origin gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloroplast sulfate permeases in a variety of species is presented.     

Quantitative assessment of heteroplasmy levels in <Emphasis Type="Italic">Senecio vulgaris</Emphasis> chloroplast DNA

Heteroplasmy in coding chloroplast DNA was only recently shown to occur and was so far not quantitatively assessed. We present a quantitative analysis of cpDNA heteroplasmy levels at a triazine-resistance determining site within and between individual Senecio vulgaris plants. Detectable levels of heteroplasmic haplotypes were observed in all tested plants. As expected, the levels of heteroplasmy vary greatly between plants. However, even within individual plants, the fraction of one haplotype may cover a range from below 1 to well over 90. Our results suggest that heteroplasmy may be a common phenomenon in S. vulgaris.Possible consequences for molecular diagnostics of chloroplast encoded traits as well as evolutionary consequences of chloroplast heteroplasmy are discussed.     

Production and characterization of intergeneric somatic hybrids between <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> and their backcrossing progenies

Alien chromosome addition lines have been widely used for identifying gene linkage groups, assigning species-specific characters to a particular chromosome and comparing gene synteny between related species. In plant breeding, their utilization lies in introgressing characters of agronomic value. The present investigation reports the production of intergeneric somatic hybrids Brassica napus (2n = 38) + Orychophragmus violaceus (2n = 24) through asymmetric fusions of mesophyll protoplasts and subsequent development of B. napus-O. violaceous chromosome addition lines. Somatic hybrids showed variations in morphology and fertility and were mixoploids (2n = 51–67) with a range of 19–28 O. violaceus chromosomes identified by genomic in situ hybridization (GISH). After pollinated with B. napus parent and following embryo rescue, 20 BC₁ plants were obtained from one hybrid. These exhibited typical serrated leaves of O. violaceus or B. napus-type leaves. All BC₁ plants were partially male fertile but female sterile because of abnormal ovules. These were mixoploids (2n = 41–54) with 9–16 chromosomes from O. violaceus. BC₂ plants showed segregations for female fertility, leaf shape and still some chromosome variation (2n = 39–43) with 2–5 O. violaceus chromosomes, but mainly containing the whole complement from B. napus. Among the selfed progenies of BC₂ plants, monosomic addition lines (2n = 39, AACC + 1O) with or without the serrated leaves of O. violaceus or female sterility were established. The complete set of additions is expected from this investigation. In addition, O. violaceus plants at diploid and tetraploid levels with some variations in morphology and chromosome numbers were regenerated from the pretreated protoplasts by iodoacetate and UV-irradiation. Z. Zhao and T. Hu make equal contributions to this work.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

<Emphasis Type="Italic">Piriformospora indica</Emphasis> and <Emphasis Type="Italic">Sebacina vermifera</Emphasis> increase growth performance at the expense of herbivore resistance in <Emphasis Type="Italic">Nicotiana attenuata</Emphasis>

A Sebacinales species was recovered from a clone library made from a pooled rhizosphere sample of Nicotiana attenuata plants from 14 native populations. Axenic cultures of the related species, Piriformospora indica and Sebacina vermifera, were used to examine their effects on plant performance. Inoculation of N. attenuata seeds with either fungus species stimulated seed germination and increased growth and stalk elongation. S. vermifera inoculated plants flowered earlier, produced more flowers and matured more seed capsules than did non-inoculated plants. Jasmonate treatment during rosette-stage growth, which slows growth and elicits herbivore resistance traits, erased differences in vegetative, but not reproductive performance resulting from S. vermifera inoculation. Total nitrogen and phosphorous contents did not differ between inoculated and control plants, suggesting that the performance benefits of fungal inoculation did not result from improvements in nutritional status. Since the expression of trypsin proteinase inhibitors (TPI), defensive proteins which confer resistance to attack from Manduca sexta larvae, incur significant growth and fitness costs for the plant, we examined the effect of S. vermifera inoculation on herbivore resistance and TPI activity. After 10 days of feeding on S. vermifera-inoculated plants, larval mass was 46% higher and TPI activity was 48% lower than that on non-inoculated plants. These results suggest that Sebacina spp. may interfere with defense signaling and allow plants to increase growth rates at the expense of herbivore resistance mediated by TPIs.     

Pathogenicity of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> and <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> to the tobacco spider mite <Emphasis Type="Italic">Tetranychus evansi</Emphasis>

Seventeen isolates of Metarhizium anisopliae (Metschnikoff) Sorokin and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated for their pathogenicity against the tobacco spider mite, Tetranychus evansi Baker & Pritchard. In the laboratory all the fungal isolates were pathogenic to the adult female mites, causing mortality between 22.1 and 82.6%. Isolates causing more than 70% mortality were subjected to dose–response mortality bioassays. The lethal concentration causing 50% mortality (LC₅₀) values ranged between 0.7×10⁷ and 2.5×10⁷ conidia ml⁻¹. The lethal time to 50% mortality (LT₅₀) values of the most active isolates of B. bassiana and M. anisopliae strains varied between 4.6 and 5.8 days. Potted tomato plants were artificially infested with T. evansi and treated with B. bassiana isolate GPK and M. anisopliae isolate ICIPE78. Both fungal isolates reduced the population density of mites as compared to untreated controls. However, conidia formulated in oil outperformed the ones formulated in water. This study demonstrates the prospects of pathogenic fungi for the management of T. evansi.     

Characterization of the cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from marine green alga, <Emphasis Type="Italic">Bryopsis corticulans</Emphasis>

A pure, active cytochrome b ₆ f was isolated from the chloroplasts of the marine green alga, Bryopsis corticulans. To investigate and characterize this cytochrome b ₆ f complex, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), absorption spectra measurement and HPLC were employed. It was shown that this purified complex contained four large subunits with apparent molecular masses of 34.8, 24, 18.7 and 16.7 kD. The ratio of Cyt b ₆ to Cytf was 2.01 : 1. The cytochromeb ₆ f was shown to catalyze the transfer of 73 electrons from decylplastoquinol to plastocyanin–ferricyanide per Cyt f per second. α-Carotene, one kind of carotenoid that has not been found to present in cytochrome b ₆ f complex, was discovered in this preparation by reversed phase HPLC. It was different from β-carotene usually found in cytochrome b ₆ f complex. The configuration of the major α-carotene component was assigned to be 9-cis by resonance Raman spectroscopy. Different from the previous reports, the configuration of this α-carotene in dissociated state was determined to be all-trans. Besides this carotene, chlorophyll a was also found in this complex. It was shown that the molecular ratios of chlorophylla, cis and all-trans-α-carotene to Cyt f in this complex were 1.2, 0.7 and 0.2, respectively.     

L-<Emphasis Type="Italic">myo</Emphasis>-inositol-1-phosphate synthase: partial purification and characterisation from <Emphasis Type="Italic">Gleichenia glauca</Emphasis>

A screening for the enzyme L-myo-inositol-1-phosphate synthase [EC 5.5.1.4] has been made first time in both vegetative and reproductive parts of the representative members of pteridophytes: Lycopodium, Selaginella, Equisetum, Polypodium, Dryopteris, and Gleichenia. The enzyme has been partially purified following low-speed centrifugation, streptomycin sulphate precipitation, ammonium sulphate fractionation, chromatography on DEAE-cellulose and gel-filtration through Sephadex G-200, and characterised from the reproductive pinnules of Gleichenia glauca Smith. The enzyme has a pH optimum at 7.5. The K_m for glucose-6-P and NAD⁺ were 0.922 × 10^–3 M and 0.9 × 10^–4 M, respectively. A basal activity of the enzyme has been recorded in absence of exogenous NAD⁺. The enzyme activity was augmented with NH₄Cl, but heavy metals like Hg²⁺, Cu²⁺ and Zn²⁺ inactivated it.     

Thermostable malate synthase of<Emphasis Type="Italic"> Streptomyces thermovulgaris</Emphasis>

The gene, encoding malate synthase (MS), aceB, was cloned from the thermophilic bacterium Streptomyces thermovulgaris by homology-based PCR. The 1,626-bp cloned fragment encodes a protein consisting of 541 amino acids. S. thermovulgaris malate synthase (stMS) gene was over-expressed in Escherichia coli using a glutathione-S transferase (GST) fusion vector (pGEX-6P-1), purified by affinity chromatography, and subsequently cleaved from its GST fusion partner. The purified stMS was characterized and compared to a mesophilic malate synthase (scMS) from Streptomyces coelicolor. stMS exhibited higher temperature optima (40–60 °C) than those of scMS (28–37 °C). It was more thermostable and very resistant to the chemical denaturant urea. Amino acid sequence comparison of stMS with four mesophilic streptomycete MSs indicated that they share 70.9–91.4% amino acid identities, with stMS possessing slightly more charged residues (~31%) than its mesophilic counterparts (~28–29%). Seven charged residues (E85, R187, R209, H239, H364, R382 and K520) that were unique to stMS may be selectively and strategically placed to support its peculiar characteristics.     

Photosynthesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Mutants with Reduced Chloroplast Number

We have used a class of Arabidopsis mutants altered in the accumulation and replication of chloroplasts (arc mutants) to investigate the effect of reduced chloroplast number on the photosynthetic competence of leaves. Each of the arc mutants examined (arc3, arc5, and arc6) accumulate only a few (2–15) large chloroplasts per mesophyll cell [K.A. Pyke and R.M. Leech (1992) Plant Physiology 99: 1005–1008]. The increased plastid size maintains a constant plastid to mesophyll cell volume, which has been suggested to compensate for the lower chloroplast number. In fact, we find that reduced chloroplast number has an effect on both the composition and structure of the photosynthetic apparatus, and that each arc mutant has an altered photosynthetic capacity, and we conclude that photosynthetic competence is dependent on proper chloroplast division and development.     

Transgenic resistance to <Emphasis Type="Italic">Cymbidium mosaic virus</Emphasis> in <Emphasis Type="Italic">Dendrobium</Emphasis> expressing the viral capsid protein gene

A Taiwan isolate of Cymbidium mosaic virus (CymMV-CS) was isolated from infected Cymbidium sinesis Willd. The cDNA of the capsid protein (CP) gene was synthesized and sequenced. Alignment of this CP gene with other reported CPs revealed homologies of 92–98% at the nucleotide level and 98–99% at the amino acid level. To generate virus-resistant varieties, the CymMV-CS CP gene was transformed into Dendrobium protocorms through particle bombardment. Transformants were selected on medium supplemented with 20 mg/L hygromycin and the presence of the transgene was confirmed by polymerase chain reaction, Southern, Northern and Western blot analyses. Transgenic Dendrobium harboring the CymMV CP gene expressed a very low level of virus accumulation four months post-inoculation with CymMV, as detected by ELISA. The transgenic plants exhibited much milder symptoms than the non-transgenic plants upon challenge with CymMV virionsSequence data reported from this article have been deposited at the GenBank Data Libraries under Accession No. AY429021.