The <Emphasis Type="Italic">rolB</Emphasis> gene promotes rooting <Emphasis Type="Italic">in vitro </Emphasis>and increases fresh root weight <Emphasis Type="Italic">in vivo</Emphasis> of transformed apple scion cultivar ‘Florina’

Transgenic apple (Malus × domestica Borkh.) Florina plants were obtained by Agrobacterium-mediated transformation. The efficiency of gene transfer was 7.9%, calculated as a number of explants producing at least one transgenic shoot, after co-cultivation of leaf explants from in vitro-grown shoots in a thin layer of the A. tumefaciens C58C1 strain with the binary vector pCMB-B:GUS. Polymerase chain reaction revealed that all the clones contained the nptII and rolB genes, while four of them did not contain the gus gene. Southern blot analysis confirmed the integration of the nptII and rolB genes, with one to three copies per genome being present. All independent rolB-transgenic lines were able to produce roots in vitro on the hormone free medium, while the plants, transformed with the vector pIB16.1, or untransformed control plants did not root, and only half of shoots of MM106 rootstock rooted on this medium. The average root number in the rolB-transgenic clones ranged from 4 to 7.7. Pretreatment with indole-3-butyric acid caused root formation in all transgenic and control plants and significantly increased root number in the rolB-transgenic lines, compared to untransformed plants. RolB-transgenic plants, grown in vivo in greenhouse for 2 years, did not differ phenotypically from the wild type line with the exception of root parts. All rolB-transformed plants produced altered root systems containing more fine roots leading to significantly increased fresh root weight in five plant lines.     

Primary and repetitive secondary somatic embryogenesis in <Emphasis Type="Italic">Rosa hybrida</Emphasis> ‘Samantha’

This study describes a plant regeneration system for Rosa hybrida ‘Samantha’, a mainstream commercial cultivar, via direct somatic embryogenesis. Somatic embryogenesis was induced from in vitro-derived leaf explants, achieving a frequency of 7.5% following 8 weeks of culture on a Murashige and Skoog (MS) medium supplemented with 3.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 30 g/L glucose. We evaluated the effects of various types and concentrations of carbohydrates and plant growth regulators (PGRs) on the proliferation and germination of secondary somatic embryos. MS medium containing 1.0 mg/L 2,4-D, 0.05 mg/L 6-benzyladenine (BA) and 60 g/L glucose was found to be the most effective in promoting the proliferation of somatic embryos. The highest germination rate (56.2%) of somatic embryos was observed on MS medium containing 1.0 mg/L Thidiazuron (TDZ) and 0.5 mg/L BA. Whole plantlets were obtained by culturing germinated shoots on rooting medium comprising 1/2-strength MS salts supplemented with 0.05 mg/L α-Naphthalene acetic acid (NAA). Plantlets grew vigorously with normal vegetative and flowering characteristics.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Identifying QTL for high-temperature adult-plant resistance to stripe rust (<Emphasis Type="Italic">Puccinia striiformis</Emphasis> f. sp. <Emphasis Type="Italic">tritici</Emphasis>) in the spring wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) cultivar ‘Louise’

Over time, many single, all-stage resistance genes to stripe rust (Puccinia striiformis f. sp. tritici) in wheat (Triticum aestivum L.) are circumvented by race changes in the pathogen. In contrast, high-temperature, adult-plant resistance (HTAP), which only is expressed during the adult-plant stage and when air temperatures are warm, provides durable protection against stripe rust. Our objective was to identify major quantitative trait loci (QTL) for HTAP resistance to stripe rust in the spring wheat cultivar ‘Louise’. The mapping population consisted of 188 recombinant inbred lines (RIL) from a Louise (resistant) by ‘Penawawa’ (susceptible) cross. F_5:6 lines were evaluated for stripe rust reaction under natural infection in replicated field trials at five locations in the US Pacific Northwest in 2007 and 2008. Infection type (IT) and disease severity were recorded for each RIL 2–4 times per location. In all environments, Penawawa, the susceptible parent, was rated with an IT ranging from 6 to 8 at all growth stages evaluated. In contrast, Louise, the resistant parent, was rated with an IT of 2 or 3 across growth stages. Distribution of IT values was bimodal, indicating a single major gene was affecting the trait. The parents and RIL population were evaluated with 295 polymorphic simple sequence repeat and one single nucleotide polymorphism markers. One major QTL, designated QYrlo.wpg-2BS, associated with HTAP resistance in Louise, was detected on chromosome 2BS (LOD scores ranging from 5.5 to 62.3 across locations and years) within a 16.9 cM region flanked by Xwmc474 and Xgwm148. SSR markers associated with QYrlo.wpg-2BS are currently being used in marker-based forward breeding strategies to transfer the target region into adapted germplasm to improve the durability of resistance in resulting cultivars.     

Deletion of <Emphasis Type="Italic">psbQ</Emphasis>’ gene in <Emphasis Type="Italic">Cyanidioschyzon merolae</Emphasis> reveals the function of extrinsic <Emphasis Type="Italic">PsbQ</Emphasis>’ in PSII

Key message

We have successfully produced single-cell colonies of C. merolae mutants, lacking the PsbQ’ subunit in its PSII complex by application of DTA-aided mutant selection. We have investigated the physiological changes in PSII function and structure and proposed a tentative explanation of the function of PsbQ’ subunit in the PSII complex.

Abstract

We have improved the selectivity of the Cyanidioschyzon merolae nuclear transformation method by the introduction of diphtheria toxin genes into the transformation vector as an auxiliary selectable marker. The revised method allowed us to obtained single-cell colonies of C. merolae, lacking the gene of the PsbQ’ extrinsic protein. The efficiency of gene replacement was extraordinarily high, allowing for a complete deletion of the gene of interest, without undesirable illegitimate integration events. We have confirmed the absence of PsbQ’ protein at genetic and protein level. We have characterized the physiology of mutant cells and isolated PSII protein complex and concluded that PsbQ’ is involved in nuclear regulation of PSII activity, by influencing several parameters of PSII function. Among these: oxygen evolving activity, partial dissociation of PsbV, regulation of dimerization, downsizing of phycobilisomes rods and regulation of zeaxanthin abundance. The adaptation of cellular physiology appeared to favorite upregulation of PSII and concurrent downregulation of PSI, resulting in an imbalance of energy distribution, decrease of photosynthesis and inhibition of cell proliferation.

     

Effects of different amino acids on somatic embryogenesis of strawberry (<Emphasis Type="Italic">Fragaria</Emphasis> × <Emphasis Type="Italic">ananassa</Emphasis> Duch.)

This study was conducted to optimize different types and concentrations of amino acids on somatic embryogenesis induction, development and maturation of leaf explants in strawberry cultivars (‘Camarosa’, ‘Paros’ and ‘Kurdistan’). Calli derived from leaf sections were transferred onto MS medium with 1.0 mg/l 2,4-d + 0.5 mg/l BAP supplemented with 0.0, 50, 100, 150 or 200 mg/l concentrations of proline, alanine and glutamine. Stimulation of embryogenesis and embryo development was strictly dependent on the type and concentration of amino acid in the medium. Proline (100 mg/l) was much more effective than glutamine and alanine, on induction and development of somatic embryogenesis in all cultivars. Cultures grown on amino acid-free medium attained lower somatic embryos than cultures grown on amino acid treated medium. Low concentrations (50 mg/l) and high concentrations (200 mg/l) of amino acids tested were inefficient for embryogenesis induction as well as for somatic embryos development.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Gibberellic acid promotes in vitro regeneration and shoot multiplication in <Emphasis Type="Italic">Lotus</Emphasis><Emphasis Type="Italic">corniculatus</Emphasis> L.

Shoots of Lotus corniculatus L., previously transformed with Agrobacterium tumefaciens LBA4404/pTOK233, were grown in gibberellic acid (GA₃)-containing media in an attempt to improve their growth and multiplication. Nodal stem segments of four poorly multiplying clones, a clone with very good multiplication, and two non-transformed clones were incubated in media containing GA₃ for 3 weeks, and then returned to a hormone-free medium for a further 3–5 subculture periods. Gibberellic acid increased the number of axillary buds from one in controls to 2–13 shoots during the first 3 weeks of subculture in GA₃-containing media. The multiplied buds, which apparently derived from additional axillaries, continued to multiply in hormone-free medium, producing bunches of more than 50 shoots after 9 weeks. In addition, many nodal segments also produced callus tissues at the basal end, or along the internode below the node, in which abundant shoot meristems formed de novo. Histological examination confirmed their origin via organogenesis. About 25% of regenerated shoots rooted spontaneously. The application of auxins significantly improved rooting in more than 75% of all clones. Well-developed plantlets were produced upon the transfer of rooted shoots to pots. No differences in responses to hormones were observed between LBA4404/pTOK233-transformed clones and non-transformed clones, indicating that the reaction to GA₃ was not the consequence of transformation events. The results point to a possible involvement of GA₃ in lateral branching. They might also be recommended as a procedure suitable for increasing the production of transformed plants in L. corniculatus.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Production of a Soluble Disulfide Bond-Linked TCR in the Cytoplasm of <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis><Emphasis Type="Italic">trx</Emphasis>B <Emphasis Type="Italic">gor</Emphasis> Mutants

Previously, we have described the use of phage display to generate high affinity disulfide bond-linked T cell receptors (TCRs). The affinities of the mutant TCRs were analysed after refolding of separately expressed α and β chains from Escherichia coli inclusion bodies. This approach is only suitable for the analysis of small numbers of TCR variants. An attractive alternative would be soluble expression within the bacterial periplasm, but the generic production of TCRs within the E. coli periplasm has so far not proved successful. Here we show that functional, soluble TCR can be produced within the cytoplasm of trxB gor mutant E. coli strains, with maximum yields of 3.4 mg/l. We also investigated the effect of coexpressing the folding modulators Skp and DsbC finding that the TCR expression levels were largely unaffected by these chaperones. Importantly, we demonstrated that the amount of protein purified from 50 ml starter cultures was sufficient to show functionality of the TCR by specific antigen binding in both ELISA and surface plasmon resonance (SPR) assays. This TCR production method has the potential to allow rapid and medium throughput analysis of affinity-matured TCRs selected from TCR phage display libraries.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

Conjugal transfer using the bacteriophage ϕC31 <Emphasis Type="Italic">att</Emphasis>/<Emphasis Type="Italic">int</Emphasis> system and properties of the <Emphasis Type="Italic">attB</Emphasis> site in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis>

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage ϕC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl₂ without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the ϕC31 att/int system.     

Direct somatic embryogenesis of potato [<Emphasis Type="Italic">Solanum tuberosum</Emphasis> (L.)] cultivar ‘Kufri Chipsona 2’

Direct somatic embryo induction was achieved from leaf and internodal explants of Solanum tuberosum (L.) cultivar ‘Kufri Chipsona 2’ on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 10.0 µM silver nitrate (MS1 medium) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 2.5 µM) and 6-benzyladenine (BA; 1.0 µΜ). It was observed that in absence of AgNO₃, friable callus was induced from cut ends of the explants, which does not develop into any kind of organised structure; thus highlighting the requirement of AgNO₃ for somatic embryogenesis in potato. Furthermore, the effect of medium strength, sucrose concentration and heat shock treatment on somatic embryogenic potential of explants was also investigated. When the strength of basal medium was reduced to half, the frequency of internodal segments differentiating somatic embryos was almost double in comparison to full strength MS medium. Sucrose concentration and heat shock treatment were found to have interactive effect on somatic embryo induction. Explants subcultured on medium containing 174 mM sucrose and subjected to heat shock (1 h; 50 °C) showed maximum somatic embryo differentiation. Although, the percent explants differentiating somatic embryos decreased sharply with increase in sucrose concentration (>?174 mM), yet the number of somatic embryos differentiated per explant were found to increase with further increase in sucrose concentration. Histological observations revealed that somatic embryos directly developed from epidermis of leaf explant and cut ends of internodal segments progressed from globular to cotyledonary stage after passing through intermediate embryogenic stages (heart shaped and torpedo shaped). Conversion of somatic embryos into plantlets (92%) was achieved on MS1 medium supplemented with BA (10.0 µM) and gibberellic acid (15.0 µM) and all regenerated plants were found to be phenotypically alike.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.