The<Emphasis Type="Italic"> HY2</Emphasis> gene as an efficient marker for transposon excision in<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Transposable elements can generate germinal and somatic mutations, and hence represent a powerful tool for the analysis of gene function. Transposons from maize have been adapted to mutagenise the genomes of diverse species. The efficiency of these systems partly relies on the ease with which germinal (i.e. germinally transmitted) or somatic excisions can be detected. Here we describe the use of HY2, a gene that codes for an enzyme involved in the biosynthesis of the phytochrome chromophore, to monitor the excision of a Ds gene-trap element in Arabidopsis thaliana. Taking advantage of the altered germination and de-etiolation behaviour of a Ds -tagged hy2 mutant, we have designed an efficient protocol for the recovery of germinal revertants, making HY2 the most precocious excision marker available, to the best of our knowledge. In addition, HY2 is also useful for generating visible sectors in photosynthetic tissues, thanks to the somatic instability of this mutable hy2 allele.Communicated by M.-A. Grandbastien     

Expression of the <Emphasis Type="Italic">HEV2.1</Emphasis> gene promoter in transgenic <Emphasis Type="Italic">Hevea brasiliensis</Emphasis>

In this article we describe the identification of endophytic bacteria belonging to three groups isolated from shoot tip cultures of banana cv. Grand Naine in a recent study (Thomas et al. 2008) based on partial 16S rRNA gene sequence homology analysis. The first group included banana stocks that displayed obvious colony growth on MS based tissue culture medium during the first in vitro passage. The second group constituted stocks that were tissue index-negative for cultivable bacteria initially but turned index-positive after a few to several (4–8) in vitro passages while the third group formed one sub-stock that turned index-positive after about 18 passages. The organisms belonged to about 20 different genera comprising of α, β, γ-proteobacteria, Gram-positive firmicutes and actinobacteria. Visibly expressing easily cultured organisms during the first in vitro passage included Enterobacter, Klebsiella, Ochrobactrum, Pantoea, Staphylococcus and Bacillus spp. Organisms of second group that were not detected or non-culturable originally constituted Brevundimonas, Methylobacterium, Alcaligenes, Ralstonia, Pseudomonas, Corynebacterium, Microbacterium, Staphylococcus, Oceanobacillus and Bacillus spp. while the third group that turned cultivable after extended in vitro culturing included mostly non-filamentous actinobacteria (Brachybacterium, Brevibacterium, Kocuria and Tetrasphaera spp.). The identification results suggested that the endophytes of second and third groups were not strictly obligate or fastidious microbes but those surviving in viable but-non-culturable (VBNC) state and displaying gradual activation to cultivable form during continuous tissue culturing. Several of the organisms isolated are known as beneficial ones in agriculture while some organisms have possible implications in human health. The use of tissue cultures for isolating uncommon endophytes is discussed. Supply of live bacterial cultures or genetic material for research purpose is subject to their revival from glycerol stocks (as some of the organisms showed poor tolerance) and the requestor obtaining written permission from the Director General, Indian Council of Agricultural Research, New Delhi-110001.     

<Emphasis Type="Italic">Rol</Emphasis> (root loci) gene as a positive selection marker to produce marker-free <Emphasis Type="Italic">Petunia hybrida</Emphasis>

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring a rol-type MAT vector, pMAT101, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing rol gene as the selection marker gene. LacZ gene was used as a model gene of interest. Infected explants were cultured on plant growth regulator (PGR)- and antibiotic-free half-strength MS medium. Sixty-five percent of the infected explants produced hairy roots. The hairy roots were separated and proliferated on 1/2 MS hormone-free medium. Shoots produced from the hairy roots on 1/2 MS medium supplemented with benzylaminopurine (BA) and naphthalene acetic acid (NAA) exhibited hairy root syndrome (Ri syndrome) such as dwarfed, reduced apical dominance, short internodes and increased rooting, but subsequently produced normal-looking marker-free shoots. Molecular analysis of DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only LacZ gene, and the removable cassette consisting of rol, R (recombinase) and GUS genes was excised. From this study it can be concluded that the chimeric rol genes can be used as a selection marker for Agrobacterinum-mediated transformation of Petunia hybrida and that the production of marker-free normal transgenic plants is possible without using selective chemical agents employing rol-type MAT vector.     

Actinomycetemcomitin: a new bacteriocin produced by <Emphasis Type="Italic">Aggregatibacter</Emphasis> (<Emphasis Type="Italic">Actinobacillus</Emphasis>) <Emphasis Type="Italic">actinomycetemcomitans</Emphasis>

Aggregatibacter (Actinobacillus) actinomycetemcomitans P_7–20 strain isolated from a periodontally diseased patient has produced a bacteriocin (named as actinomycetemcomitin) that is active against Peptostreptococcus anaerobius ATCC 27337. Actinomycetemcomitin was produced during exponential and stationary growth phases, and its amount decreased until it disappeared during the decline growth phase. It was purified by ammonium sulphate precipitation (30–60% saturation), and further by FPLC (mono-Q ionic exchange and Phenyl Superose hydrophobic interaction) and HPLC (C-18 reversed-phase). This bacteriocin loses its activity after incubation at a pH below 7.0 or above 8.0, following heating for 30 min at 45°C, and after treatment with proteolytic enzymes such as trypsin, α-chymotrypsin, and papain. Actinomycetemcomitin has a molecular mass of 20.3 KDa and it represents a new bacteriocin from A. actinomycetemcomitans.     

Transformation of<Emphasis Type="Italic"> Kalanchoe blossfeldiana</Emphasis> with <Emphasis Type="Italic">rol-</Emphasis>genes is useful in molecular breeding towards compact growth

Dwarf genotypes of the economically important flowering potted plant Kalanchoe blossfeldiana were developed by molecular breeding. Root inducing (Ri)-lines were regenerated by applying CPPU to the hairy roots, which were produced by inoculating leaf explants with a wild-type Agrobacterium rhizogenes strain ATCC15834. Amplification by polymerase chain reaction (PCR) and Southern blot analysis confirmed the presence of T-DNA in the Ri-lines. Six Ri-lines were characterised in a greenhouse trial revealing that several morphological traits changed with respect to ornamental value such as plant height, number of lateral shoots, leaf size, leaf number, flower size and number of flowers. The Ri-lines differed in their degree of Ri-phenotype, and the internodes of the Ri-lines were clearly shorter, giving a compact growth habit compared to control plants. Time to anthesis was the same in Ri-line 331 as in control plants and delayed by only 3 days in Ri-line 306 as compared to control plants. A compact plant without delayed flowering can be assumed to be valuable for further breeding.     

Transient expression of heterologous model gene in plants using <Emphasis Type="Italic">Potato virus</Emphasis><Emphasis Type="Italic">X</Emphasis>-based vector

To optimize the efficiency of expression of foreign proteins using Potato virus X (PVX) -- based vector, the gene for the coat protein (CP) of other virus (Potato virus A, PVA) was cloned into the vector, propagated in E. coli and subsequently inoculated or agroinfected into the host plants. Host range studies showed that the best host plant is N. benthamiana. By means of RT PCR the presence and the stability of the construct were tested. Both ELISA and Western blot analysis were applicable for expressed protein detection. Expression level of PVA CP achieved approximately 5--10 per mille of total soluble proteins. The results demonstrated that agroinfection is the most suitable method for the propagation of our model gene using PVX--based vectors.     

Cre/<Emphasis Type="Italic">lox</Emphasis> system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect

A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.     

Methyl jasmonate stimulates the formation and the accumulation of anthocyanin in <Emphasis Type="Italic">Kalanchoe blossfeldiana</Emphasis>

Methyl jasmonate (JA-Me) at concentrations of 0.1, 0.5 and 1.0 % (w/w) greatly stimulated anthocyanins accumulation in shoots of young plants of Kalanchoe blossfeldiana when it was applied around the stem as a lanolin paste. Stimulatory effect of JA-Me was evidently observed as early as two days after treatment. Anthocyanins were formed in the main and lateral shoots, including petioles, both below and above portions of the treatment. When leaves were removed from the plant, almost no anthocyanin formation was observed. It should be mentioned that leaves are necessary for the anthocyanin accumulation in stems induced by JA-Me.     

Expression of active <Emphasis Type="Italic">Streptomyces</Emphasis> phage phiC31 integrase in transgenic wheat plants

Site-specific recombination systems are becoming an important tool for the genetic modification of crop plants. Here we report the functional expression of the Streptomyces phage-derived phiC31 recombinase (integrase) in wheat. T-DNA constructs containing a phiC31 integrase transgene were stably transformed into wheat plants via particle gun bombardment. A plant-virus-based assay system was used to monitor the site-specific recombination activity of the recombinant integrase protein in vivo. We established several independent doubled haploid (DH) inbred lines that constitutively express an active integrase enzyme without any apparent detrimental effects on plant growth and development. The potential of phiC31 integrase expression in crop plants related to transgene control technologies or hybrid breeding systems is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. M. Rubtsova and K. Kempe contributed equally to the paper.     

Evidence for positive selection in the gene <Emphasis Type="Italic">fruitless</Emphasis> in <Emphasis Type="Italic">Anastrepha</Emphasis>fruit flies

Background  

Many genes involved in the sex determining cascade have indicated signals of positive selection and rapid evolution across different species. Even though fruitless is an important gene involved mostly in several aspects of male courtship behavior, the few studies so far have explained its high rates of evolution by relaxed selective constraints. This would indicate that a large portion of this gene has evolved neutrally, contrary to what has been observed for other genes in the sex cascade.     

Analysis of the <Emphasis Type="Italic">MAT1-2-1</Emphasis> gene of <Emphasis Type="Italic">Colletotrichum lindemuthianum</Emphasis>

A single MAT1-2-1 gene was identified from a mating pair of the filamentous ascomycete Colletotrichum lindemuthianum. The MAT1-2-1 genes from both mating partners carried an open reading frame (ORF) of 870 bp encoding a putative protein of 290 amino acids that includes the highly conserved high mobility group (HMG) domain typical of the fungal MAT1-2-1 genes. Three introns were confirmed within the C. lindemuthianum ORF, two of which were found to be conserved relative to a previously reported MAT1-2-1 gene from C. gloeosporioides. The amino acid sequence of the HMG domain from C. lindemuthianum MAT1-2-1 was also compared with those from other ascomycetes. These results suggest that although the MAT1-2-1 genes are highly conserved among ascomycetes, the mechanism which defines mating partners in the genus Colletotrichum is distinct to the idiomorph system described for other members of this phylum.     

High-resolution mapping and gene prediction of <Emphasis Type="Italic">Xanthomonas Oryzae</Emphasis> pv. <Emphasis Type="Italic">Oryzae</Emphasis> resistance gene <Emphasis Type="Italic">Xa7</Emphasis>

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a devastating disease in rice worldwide. The resistance gene Xa7, which provides dominant resistance against the pathogen with avirulence (Avr) gene AvrXa7, has proved to be durably resistant to BB. A set of SSR markers were selected from the “gramene” database based on the Xa7 gene initial mapping region on chromosome 6. These markers were used to construct a high-resolution genetic map of the chromosomal region surrounding the Xa7 gene. An F₂ mapping population with 721 highly susceptible individuals derived from a cross between the near isogenic lines (NILs) IRBB7 and IR24 were constructed to localize the Xa7 gene. In a primary analysis with eleven polymorphic SSR markers, Xa7 was located in approximately the 0.28-cM region. To walk closer to the target gene, recombinant F₂ individuals were tested using newly developed STMS (sequence tagged microsatellite) markers. Finally, the Xa7 gene was mapped to a 0.21-cM interval between the markers GDSSR02 and RM20593. The Xa7-linked markers were landed on the reference sequence of cv. Nipponbare through bioinformatics analysis. A contig map corresponding to the Xa7 gene was constructed. The target gene was assumed to span an interval of approximately 118.5-kb which contained a total of fourteen genes released by the TIGR Genome Annotation Version 5.0. Candidate-gene analysis of Xa7 revealed that the fourteen genes encode novel domains that have no amino acid sequence similar to other cloned Xa(xa) genes. Shen Chen and Zhanghui Huang are contributed equally to this work.     

Genetic tools for <Emphasis Type="Italic">Sulfolobus</Emphasis> spp.: vectors and first applications

Sulfolobus species belong to the best-studied archaeal organisms but have lacked powerful genetic methods. Recently, there has been considerable progress in the field of Sulfolobus genetics. Urgently needed basic genetic tools, such as targeted gene knockout techniques and shuttle vectors are being developed at an increasing pace. For S. solfataricus knockout systems as well as different shuttle vectors are available. For the genetically more stable S. acidocaldarius shuttle vectors have been recently developed. In this review we summarize the currently available genetic tools and methods for the genus Sulfolobus. Different transformation protocols are discussed, as well as all so far developed knockout systems and Sulfolobus-Escherichia coli shuttle vectors are summarized. Special emphasis is put on the important vector components, i.e., selectable markers and Sulfolobus replicons. Additionally, the information gathered on different Sulfolobus strains with respect to their use as recipient strains is reviewed. The advantages and disadvantages of the different systems are discussed and aims for further improvement of genetic systems are identified.     

Studies on genetic transformation of olive (<Emphasis Type="Italic">Olea europaea</Emphasis> L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for <Emphasis Type="Italic">nptII</Emphasis> selection; II. Transient transformation via particle bombardment

As a first step to the establishment of a genetic transformation protocol for olive somatic embryos obtained from the seeds of cv. ‘Picual’, the efficiencies of different aminoglycoside antibiotics as selective agents to be used with the nptII marker gene, and the particle bombardment technique for transient transformation have been evaluated. Among the three antibiotics tested, paromomycin and kanamycin showed a similar inhibitory effect and, at 200 mg l⁻¹, both of them impaired callus growth after 8 weeks of culture. However, when isolated embryos were cultured in the presence of these antibiotics, a 20% of the embryos still remained viable at 400 mg l⁻¹. Neomycin was discarded as a selective agent since it showed only a moderate toxic effect. Contrary to solid medium, when olive callus was cultured in liquid medium supplemented with different paromomycin concentrations for 3 weeks, the callus growth was impaired at the lowest antibiotic concentration, 3 mg l⁻¹. Best conditions for transient transformation of olive callus using PDS-1000/He system were a 6 cm target distance and a 900 psi bombardment pressure. pCGU∆1 plasmid, containing the gus gene under the control of sunflower ubiquitin promoter yielded a significantly higher number of gus expression areas per bombarded explant than pGUSINT or pJGUS5 plasmids, where the gus gene is driven by CaMV35S promoter or CaMV35S with enhancer, respectively. Almost 45% of bombarded explants showed gus expression 12 weeks after bombardment.     

The expression of analgesic-antitumor peptide (<Emphasis Type="Italic">AGAP</Emphasis>) from Chinese <Emphasis Type="Italic">Buthus</Emphasis><Emphasis Type="Italic">martensii</Emphasis> Karsch in transgenic tobacco and tomato

The present study aimed to obtain analgesic-antitumor peptide (AGAP) gene expression in plants. The analgesic-antitumor peptide (AGAP) gene was from the venom of Buthus martensii Karsch. Previous studies showed that AGAP has both analgesic and antitumor activities, suggesting that AGAP would be useful in clinical situations as an antitumor drug. Given that using a plant as an expression vector has more advantages than prokaryotic expression, we tried to obtain transgenic plants containing AGAP. In the present study, the AGAP gene was cloned into the plasmid pBI121 to obtain the plant expression vector pBI-AGAP. By tri-parental mating and freeze–thaw transformation, pBI-AGAP was transformed into Agrobacterium tumefaciens LBA4404. Tobacco (Nicotiana tabacum) and tomato (Lycopersicom esculentum) were transformed by the method of Agrobacterium-mediated leaf disc transformation. The transformants were then screened to grow and root on media containing kanamycin. Finally, transformations were confirmed by analysis of PCR, RT-PCR and western blotting. The results showed that the AGAP gene was integrated into the genomic DNA of tobacco and tomato and was successfully expressed. Therefore, the present study suggests a potential industrial application of AGAP expressed in plants.     

Production of transgenic <Emphasis Type="Italic">Pinus armandii</Emphasis> plants harbouring <Emphasis Type="Italic">btCry</Emphasis>III(<Emphasis Type="Italic">A</Emphasis>) gene

A synthetic chimeric gene SbtCryIII(A) encoding the insecticidal protein btCryIII(A), was transformed into Pinus armandii embryos and embryogenic calli using Agrobacterium tumefaciens. Polymerase chain reaction and genomic DNA Southern blot analysis showed that the SbtCryIII(A) gene was integrated into the genome of transgenic Pinus armandii plants, and Northern blot analysis indicated that the SbtCryIII(A) gene was transcribed.