Spontaneous spectinomycin resistance mutations of the chloroplast <Emphasis Type="Italic">rrn16</Emphasis> gene in <Emphasis Type="Italic">Daucus carota</Emphasis> callus lines

Bioballistic transformation of carrot (Daucus carota L.) callus cultures with a plasmid containing the aadA (aminoglycoside 3′-adenyltransferase) gene and subsequent selection of transformants on a selective medium containing spectinomycin (100–500 mg/l) yielded ten callus lines resistant to this antibiotic. PCR analysis did not detect exogenous DNA in the genomes of spectinomycin-resistant calluses. Resistance proved to be due to spontaneous mutations that occurred in two different regions of the chloroplast rrn16 gene, which codes for the 16S rRNA. Six lines displayed the G > T or G > C transverions in position 1012 of the rrn16 gene, and three lines had the A > G transition in position 1138 of the gene. Chloroplast mutations arising during passages of callus cultures in the presence of spectinomycin were described in D. carota for the first time. The cause of spectinomycin resistance was not identified in one line. The mutations observed in the D. carota plastid genome occurred in the region that is involved in the formation of a double-stranded region at the 3′ end of the 16S rRNA and coincided in positions with the nucleotide substitutions found in spectinomycin-resistant plants of tobacco Nicotiana tabacum L. and bladderpod Lesquerella fendleri L.     

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 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.     

Phosphinothricin-resistant <Emphasis Type="Italic">Brassica napus</Emphasis> + <Emphasis Type="Italic">Orychophragmus violaceus</Emphasis> somatic hybrids

Hybrid plants resistant to phosphinothricin (PPT) are obtained as a result of experiments with somatic hybridization between Brassica napus L. cv. Kalinins’kyy and Orychophragmus violaceus L. O.E. Shulz. The hybrids inherited PPT resistance from O. violaceus plants that had been previously transformed by a vector containing the maize transposon system Spm/dSPm with bar gene located within the nonautonomous transposon. The morphologically obtained plants occupy an intermediate position between the initial forms, which is in agreement with the results of isoenzyme analyses (analysis of multiple forms of amylase and esterase) and PCR analysis (presence of the genes bar, gus, and SpmTPase). Inheritance of the plastome occurs from oilseed rape, while that of the mitochondrion, from O. violaceus, which is proved by means of PCR-RFLP analysis. The plant hybrids may be utilized for further selection research with oilseed rape following determination of the edible quality of its oil as well as in experiments with chloroplast transformation, a topic which is of critical importance for oilseed rape.     

Plastid Transformation in Lesquerella Fendleri,an Oilseed Brassicacea

A plastid transformation protocol was developed for Lesquerella fendleri, a species with a high capacity for plant regeneration in tissue culture. Transformation vector pZS391B carried an aadA16gfp marker gene conferring streptomycin–spectinomycin resistance and green fluorescence under UV light. Biolistic transformation of 51 Lesquerella leaf samples, followed by spectinomycin selection, yielded two transplastomic clones. The AAD–GFP fusion protein, the marker gene product, was localized to chloroplasts by confocal laser microscopy. Fertile plants and seed progeny were obtained in line Lf-pZS391B-1. In the 51 samples a large number (108) of spontaneous mutants were identified. In five of the lines spectinomycin resistance was localized to a conserved stem structure by sequencing 16S rRNA genes. Success in L. fendleri, a wild oilseed species, extends plastid transformation beyond Arabidopsis thaliana in the Brassicaceae family.     

Stable Plastid Transformation in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes—aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)—in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.     

Plastid transformation in eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

We have developed a method for plastid transformation in eggplant (Solanum melongena L.), a solanaceous plant species. Plastid transformation in eggplant was achieved by bombardment of green stem segments with pPRV111A plastid expression vector carrying the aadA gene encoding aminoglycoside 3′′-adenylyltransferase. Biolistic delivery of the pPRV111A plasmid yielded transplastomic plants at a frequency of two per 21 bombarded plates containing 25 stem explants each. Integration of the aadA gene in the plastome was verified by PCR analysis and also by Southern blotting using 16S rDNA (targeting sequence) and the aadA gene as a probe. Transplastomic expression of the aadA gene was verified by RT-PCR. The development of transplastomic technology in eggplant may open up exciting possibilities for novel gene introduction and expression in the engineered plastome for agronomic or pharmaceutical traits.     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

Simple and efficient plastid transformation system for the liverwort <Emphasis Type="Italic">Marchantia polymorpha</Emphasis> L. suspension-culture cells

We have established a simple and efficient plastid transformation system for liverwort, Marchantia polymorpha L., suspension-culture cells, which are homogenous, chloroplast-rich and␣rapidly growing. Plasmid pCS31 was constructed to integrate an aadA expression cassette for spectinomycin-resistance into the trnI–trnA intergenic region of the liverwort plastid DNA by homologous recombination. Liverwort suspension-culture cells were bombarded with pCS31-coated gold projectiles and selected on a medium containing spectinomycin. Plastid transformants were reproducibly isolated from the obtained spectinomycin-resistant calli. Selection on a sucrose-free medium greatly improved the efficiency of selection of plastid transformants. Homoplasmic plastid transformant lines were established by␣successive subculturing for 14 weeks or longer on the spectinomycin-containing medium. The plastid transformation system of liverwort suspension-culture cells should facilitate the investigation of the fundamental genetic systems of plastid DNA, such as replication.     

Altered expression of the<Emphasis Type="Italic"> Arabidopsis</Emphasis> ortholog of<Emphasis Type="Italic"> DCL</Emphasis> affects normal plant development

The DCL (defective chloroplasts and leaves) gene of tomato (Lycopersicon esculentum Mill.) is required for chloroplast development, palisade cell morphogenesis, and embryogenesis. Previous work suggested that DCL protein is involved in 4.5S rRNA processing. The Arabidopsis thaliana (L.) Heynh. genome contains five sequences encoding for DCL-related proteins. In this paper, we investigate the function of AtDCL protein, which shows the highest amino acid sequence similarity with tomato DCL. AtDCL mRNA was expressed in all tissues examined and a fusion between AtDCL and green fluorescent protein (GFP) was sufficient to target GFP to plastids in vivo, consistent with the localization of AtDCL to chloroplasts. In an effort to clarify the function of AtDCL, transgenic plants with altered expression of this gene were constructed. Deregulation of AtDCL gene expression caused multiple phenotypes such as chlorosis, sterile flowers and abnormal cotyledon development, suggesting that this gene is required in different organs. The processing of the 4.5S rRNA was significantly altered in these transgenic plants, indicating that AtDCL is involved in plastid rRNA maturation. These results suggest that AtDCL is the Arabidopsis ortholog of tomato DCL, and indicate that plastid function is required for normal plant development.Abbreviations DCL Defective chloroplasts and leaves - GFP Green fluorescent protein     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the dwarf pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L. var. <Emphasis Type="Italic">nana</Emphasis>)

The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid (NAA), 5 μM N⁶-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed in T₁ generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment of this transformation system is invaluable for investigating fruit-tree-specific phenomena.     

<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.     

Analysis of the meiotic recombination frequency in transgenic tomato hybrids expressing <Emphasis Type="Italic">recA</Emphasis> and <Emphasis Type="Italic">NLS-recA-licBM3</Emphasis> genes

To study and induce meiotic recombination in plants, we generated and analyzed transgenic tomato hybrids F₁-RecA and F₁-NLS-recA-LicBM3 expressing, respectively, the recA gene of Escherichia coli and the NLS-recA-licBM3 gene. It was found that the recA and NLS-recA-licBM3 genes are inherited through the maternal and paternal lineages, they have no selective influence on the pollen and are contained in tomato F₁-RecA and F₁-NLS-RecA-LicBM3 hybrids outside the second chromosome in the hemizygous state. The comparative analysis of the meiotic recombination frequency (rf) in the progenies of the transgenic and nontransgenic hybrids showed that only the expression of the recA gene of E. coli in cells of the F1-RecA plants produced a 1.2–1.5-fold increase in the frequency of recombination between some linked marker genes of the second chromosome of tomato.     

Pollen-Specific Expression of <Emphasis Type="Italic">Oryza sativa Indica</Emphasis> Pollen Allergen Gene (<Emphasis Type="Italic">OSIPA</Emphasis>) Promoter in Rice and <Emphasis Type="Italic">Arabidopsis</Emphasis> Transgenic Systems

Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased. The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other crop plants.     

Molecular Systematic Study of <Emphasis Type="Italic">Chrysosplenium</Emphasis> Series <Emphasis Type="Italic">Pilosa</Emphasis> (Saxifragaceae) in Korea

To address systematic issues pertaining to series Pilosa of Korean Chrysosplenium, the phylogenetic relationships of the 35 accessions of Korean Chrysosplenium taxa were examined using the DNA sequence data obtained from the nuclear ribosomal internal transcribed spacer (ITS) regions and the psbK-I gene of the plastid genome. The results from both ITS and psbK-I sequence data indicated that Chrysosplenium flaviflorum formed a well-supported clade that merits species status of the taxon. The ITS phylogenetic trees detected two distinct lineages in Chrysosplenium pilosum var. fulvum. Geographically, one lineage was widely distributed throughout the country whereas the other was confined to Gyeonggi and Gangwon provinces. The plants sampled from Jeju Island, which have been recognized as C. pilosum var. sphaerospermum [C. hallaisanense] exhibited no DNA sequence feature segregating them from C. pilosum var. fulvum [C. barbatum] distributed in the mainland area of Korea. The plastid DNA sequence data was not fully in concordance with the nuclear ribosomal ITS data and the current taxa delimitation. These results suggest incomplete lineage sorting or that plastid genome capture may have occurred during the evolution of some taxa examined in the present study.     

Characterization of directly transformed weedy <Emphasis Type="Italic">Brassica rapa</Emphasis> and introgressed <Emphasis Type="Italic">B. rapa</Emphasis> with Bt <Emphasis Type="Italic">cry1Ac</Emphasis> and <Emphasis Type="Italic">gfp</Emphasis> genes

Crop to weed transgene flow, which could result in more competitive weed populations, is an agricultural biosafety concern. Crop Brassica napus to weedy Brassica rapa hybridization has been extensively characterized to better understand the transgene flow and its consequences. In this study, weedy accessions of B. rapa were transformed with Bacillus thuringiensis (Bt) cry1Ac- and green fluorescence protein (gfp)-coding transgenes using Agrobacterium to assess ecological performance of the wild biotype relative to introgressed hybrids in which the transgenic parent was the crop. Regenerated transgenic B. rapa events were characterized by progeny analysis, Bt protein enzyme-linked immunosorbent assay (ELISA), Southern blot analysis, and GFP expression assay. GFP expression level and Bt protein concentration were significantly different between independent transgenic B. rapa events. Similar reproductive productivity was observed in comparison between transgenic B. rapa events and B. rapa × B. napus introgressed hybrids in greenhouse and field experiments. In the greenhouse, Bt transgenic plants experienced significantly less herbivory damage from the diamondback moth (Plutella xylostella). No differences were found in the field experiment under ambient, low, herbivore pressure. Directly transformed transgenic B. rapa plants should be a helpful experimental control to better understand crop genetic load in introgressed transgenic weeds.     

In vivo effects of <Emphasis Type="Italic">NbSiR</Emphasis> silencing on chloroplast development in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Sulfite reductase (SiR) performs dual functions, acting as a sulfur assimilation enzyme and as a chloroplast (cp-) nucleoid binding protein. In this study, we examined the in vivo effects of SiR deficiency on chloroplast development in Nicotiana benthamiana. Virus-induced gene silencing of NbSiR resulted in leaf yellowing and growth retardation phenotypes, which were not rescued by cysteine supplementation. NbSiR:GFP fusion protein was targeted to chloroplasts and colocalized with cp-nucleoids. Recombinant full-length NbSiR protein and the C-terminal half of NbSiR possessed cp-DNA compaction activities in vitro, and expression of full-length NbSiR in E. coli caused condensation of genomic DNA. NbSiR silencing differentially affected expression of plastid-encoded genes, inhibiting expression of several genes more severely than others. In the later stages, depletion of NbSiR resulted in chloroplast ablation. In NbSiR-silenced plants, enlarged cp-nucleoids containing an increased amount of cp-DNA were observed in the middle of the abnormal chloroplasts, and the cp-DNAs were predominantly of subgenomic sizes based on pulse field gel electrophoresis. The abnormal chloroplasts developed prolamellar body-like cubic lipid structures in the light without accumulating NADPH:protochlorophyllide oxidoreductase proteins. Our results suggest that NbSiR plays a role in cp-nucleoid metabolism, plastid gene expression, and thylakoid membrane development.     

In Vivo PCR-DGGE Analysis of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Oenococcus oeni</Emphasis> Populations in Red Wine

In order to monitor Lactobacillus plantarum and Oenococcus oeni in red wine produced with Italian grape (variety “Primitivo di Puglia”), a polymerase chain reaction– denaturing gradient gel electrophoresis (PCR-DGGE) approach using the rpoB as gene target was established. Wine was treated or not with potassium metabisulphite and supplemented with a commercial bacterial starter of O. oeni to encourage malolactic fermentation. Samples were taken from the vinification tanks at 4, 10, 16, 22, and 28 days after the start of alcoholic fermentation. Genomic DNA was directly isolated from wine and identification of lactic acid bacteria was performed using primers rpoB1, rpoB1O, and rpoB2 able to amplify a region of 336 bp corresponding to the rpoB gene. Amplified fragments were separated in a 30–60% DGGE gradient, and the ability of the PCR-DGGE analysis to distinguish L. plantarum and O. oeni was assessed. The results reported suggest that the PCR-DGGE method, based on the rpoB gene as molecular marker, is a reproducible and suitable tool and may be of great value for wine makers in order to monitor spoilage microorganisms during wine fermentation.     

Functional characterization of the pollen-specific <Emphasis Type="Italic">SBgLR</Emphasis> promoter from potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.)

SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S. berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Zhihong Lang and Peng Zhou contributed equally to this work.