Analysis of transitive RNA silencing after grafting in transgenic plants with the coat protein gene of <Emphasis Type="Italic">Sweet potato feathery mottle virus</Emphasis>

We have previously reported the graft transmission of target specificity for RNA silencing using transgenic Nicotiana benthamiana plants expressing the coat protein gene (CP, including the 3′ non-translated region) of Sweet potato feathery mottle virus. Transgenic plants carrying the 5′ 200 and 400 bp regions of CP were newly produced. From these plants, two silenced and two non-silenced lines were selected to investigate the manifestation of transitive RNA silencing by graft experiments. Non-silenced scions carrying the entire transgene were grafted onto either 5′ or 3′ silencing inducer rootstocks. When non-silenced scions were grafted onto 5′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions and spread toward the 3′ region of the transgene mRNA. Similarly, when non-silenced scions were grafted onto 3′ silencing inducer rootstocks, RNA silencing was induced in the non-silenced scions, but was restricted to the 3′ region of the transgene and did not spread to the 5′ region. In addition, results from crossing experiments, involving non-silenced and 3′ silencing inducer plants, confirmed the above finding. This indicates that RNA silencing spreads in the 5′–3′ direction, not in the 3′–5′ direction, along the transgene mRNA.     

<Emphasis Type="Italic">Soybean dwarf virus</Emphasis>-resistant transgenic soybeans with the sense coat protein gene

We transformed a construct containing the sense coat protein (CP) gene of Soybean dwarf virus (SbDV) into soybean somatic embryos via microprojectile bombardment to acquire SbDV-resistant soybean plants. Six independent T(0) plants were obtained. One of these transgenic lines was subjected to further extensive analysis. Three different insertion patterns of Southern blot hybridization analysis in T(1) plants suggested that these insertions introduced in T(0) plants were segregated from each other or co-inherited in T(1) progenies. These insertions were classified into two types, which overexpressed SbDV-CP mRNA and accumulated SbDV-CP-specific short interfering RNA (siRNA), or repressed accumulation of SbDV-CP mRNA and siRNA by RNA analysis prior to SbDV inoculation. After inoculation of SbDV by the aphids, most T(2) plants of this transgenic line remained symptomless, contained little SbDV-specific RNA by RNA dot-blot hybridization analysis and exhibited SbDV-CP-specific siRNA. We discuss here the possible mechanisms of the achieved resistance, including the RNA silencing.     

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.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of poinsettia, <Emphasis Type="Italic">Euphorbia pulcherrima</Emphasis>, with virus-derived hairpin RNA constructs confers resistance to <Emphasis Type="Italic">Poinsettia mosaic virus</Emphasis>

Agrobacterium-mediated transformation for poinsettia (Euphorbia pulcherrima Willd. Ex Klotzsch) is reported here for the first time. Internode stem explants of poinsettia cv. Millenium were transformed by Agrobacterium tumefaciens, strain LBA 4404, harbouring virus-derived hairpin (hp) RNA gene constructs to induce RNA silencing-mediated resistance to Poinsettia mosaic virus (PnMV). Prior to transformation, an efficient somatic embryogenesis system was developed for poinsettia cv. Millenium in which about 75% of the explants produced somatic embryos. In 5 experiments utilizing 868 explants, 18 independent transgenic lines were generated. An average transformation frequency of 2.1% (range 1.2-3.5%) was revealed. Stable integration of transgenes into the poinsettia nuclear genome was confirmed by PCR and Southern blot analysis. Both single- and multiple-copy transgene integration into the poinsettia genome were found among transformants. Transgenic poinsettia plants showing resistance to mechanical inoculation of PnMV were detected by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Northern blot analysis of low molecular weight RNA revealed that transgene-derived small interfering (si) RNA molecules were detected among the poinsettia transformants prior to inoculation. The Agrobacterium-mediated transformation methodology developed in the current study should facilitate improvement of this ornamental plant with enhanced disease resistance, quality improvement and desirable colour alteration. Because poinsettia is a non-food, non-feed plant and is not propagated through sexual reproduction, this is likely to be more acceptable even in areas where genetically modified crops are currently not cultivated.     

Identification of a <Emphasis Type="Italic">Lotus</Emphasis> viral pathogen

A virus collection was used to identify a pathogen suitable for laboratory use with the model legume Lotus japonicus. Several Lotus species or L. japonicus accessions were tested and various degrees of susceptibility to the Arabis mosaic virus derived from barley (ArMV-ba) were found. Virus multiplication and persistence in Lotus tissue were examined, as well as plant responses to it. Sensitivity to the virus among the accessions and species is discussed in light of their geographical origin. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Virus-induced silencing of <Emphasis Type="Italic">Comt</Emphasis>, p<Emphasis Type="Italic">Amt</Emphasis> and <Emphasis Type="Italic">Kas</Emphasis> genes results in a reduction of capsaicinoid accumulation in chili pepper fruits

Capsaicinoids are responsible for the pungent taste of chili pepper fruits of Capsicum species. Capsaicinoids are biosynthesized through both the phenylpropanoid and the branched-fatty acids pathways. Fragments of Comt (encoding a caffeic acid O-methyltransferase), pAmt (a putative aminotransferase), and Kas (a β-keto-acyl-[acyl-carrier-protein] synthase) genes, that are differentially expressed in placenta tissue of pungent chili pepper, were individually inserted into a Pepper huasteco yellow veins virus (PHYVV)-derived vector to determine, by virus-induced gene silencing, irrespective of whether these genes are involved in the biosynthesis of capsaicinoids. Reduction of the respective mRNA levels as well as the presence of related siRNAs confirmed the silencing of these three genes. Morphological alterations were evident in plants inoculated with PHYVV::Comt and PHYVV::Kas constructs; however, plants inoculated with PHYVV::pAmt showed no evident alterations. On the other hand, fruit setting was normal in all cases. Biochemical analysis of placenta tissues showed that, indeed, independent silencing of all three genes led to a dramatic reduction in capsaicinoid content in the fruits demonstrating the participation of these genes in capsaicinoid biosynthesis. Using this approach it was possible to generate non-pungent chili peppers at high efficiency.     

Genetic transformation of <Emphasis Type="Italic">Agave salmiana</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and particle bombardment

Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive with the GUS assay after 14 months on selective medium while still undergoing regeneration.     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

Genetic transformation of golden pothos (<Emphasis Type="Italic">Epipremnum aureum</Emphasis>) mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

To establish a procedure for Agrobacterium tumefaciens-mediated transformation of golden pothos (Epipremnum aureum) plants, the effects of selection antibiotics and the preculture period of stem explants before A. tumefaciens infection were examined. Explants were co-cultivated with A. tumefaciens EHA105, harboring the plasmid pGWB2/cGUS, on a somatic embryo-inducing medium supplemented with acetosyringone. Resulting transgenic somatic embryos were screened on an antibiotic selection medium, and the transgenic pothos plants were regenerated on a germination medium. Hygromycin was the optimum selection antibiotic tested. The preculture period significantly affected the transformation efficiency, with explants precultured for one-day showing the best efficiency (5–30%). Both transformed hygromycin-resistant embryos and regenerated plants showed β-glucuronidase activity. Southern blot analysis confirmed transgene integration into the pothos genome. This reproducible transformation system for golden pothos may enable the molecular breeding of this very common indoor plant.     

Transformation of <Emphasis Type="Italic">PttKN1</Emphasis> gene to cockscomb

We have established a shoot regeneration system and genetic transformation of cockscomb (Celosia cristata and Celosia plumosus). The best results in terms of frequency of shoot regeneration and number of shoot buds per explant are observed on media supplemented with 0.5 mg l⁻¹ 6-BA (for explants of apical meristems of C. cristata) or 2.0 mg l⁻¹ 6-BA, 0.5  mg l⁻¹ NAA and 0.5  mg l⁻¹ IAA (for hypocotyls explants of C. plumosus). We use apical meristems of C. cristata and hypocotyls of C. plumosus as the starting material for transformation. A novel KNOTTED1-like homeobox1 (KNOX), PttKN1 (Populus tremula × P. tremuoides knotted1) isolated from the vascular cambial region of hybrid aspen, is introduced into cockscomb by Agrobacterium. A series of novel phenotypes are obtained from the transgenic cockscomb plants, including lobed or rumpled leaves, partite leaves and two or three leaves developed on the same petiole, on the basis of their leaf phenotypes. Transformants are selected by different concentrations of kanamycin. Transformants are confirmed by PCR of the NptII gene and PCR or RT-PCR of PttKN1 gene. Furthermore, RT-PCR shows that 35S:: PttKN1 RNA levels do not correlate with phenotypic severity. It is discussed that our results bring elements on possible function of PttKN1 gene. To our knowledge, genetic transformation of cockscomb is first reported.     

In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

Mutational analysis of the <Emphasis Type="Italic">gum</Emphasis> gene cluster required for xanthan biosynthesis in <Emphasis Type="Italic">Xanthomonas</Emphasis><Emphasis Type="Italic">oryzae</Emphasis> pv <Emphasis Type="Italic">oryzae</Emphasis>

Genome sequence analysis of Xanthomonas oryzae pv. oryzae has revealed a cluster of 12 ORFs that are closely related to the gum gene cluster of Xanthomonas campestris pv. campestris. The gum gene cluster of X. oryzae encodes proteins involved in xanthan production; however, there is little experimental evidence supporting this. In this study, biochemical analyses of xanthan produced by a defined set of X. oryzae gum mutant strains allowed us to preliminarily assign functions to most of the gum gene products: biosynthesis of the pentasaccharide repeating unit for GumD, GumM, GumH, GumK, and GumI, xanthan polymerization and transport for GumB, GumC, GumE, and GumJ, and modification of the pentasaccharide repeating unit for GumF, GumG, and GumL. In addition, we found that the exopolysaccharides are essential but not specific for the virulence of X. oryzae. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sang-Yoon Kim and Jeong-Gu Kim contributed equally to this work.     

Asymmetric somatic hybridization between UV-irradiated <Emphasis Type="Italic">Citrus unshiu</Emphasis> and <Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">sinensis</Emphasis>: regeneration and characterization of hybrid shoots

In the present paper, attempts were made to explore the possibility of employing ultraviolet (UV) irradiation in citrus asymmetric fusion for transfer of limited amount of favorable traits from a desirable cultivar to a target one. Exposure of Satsuma mandarin (Citrus unshiu Marc.) embryogenic protoplasts to UV at an intensity of 300 μW cm⁻² led to reduced viability, especially under long irradiation duration. The protoplasts could not grow during culture when they were irradiated for over 30 s. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay revealed extensive DNA fragmentation in the UV-irradiated protoplasts compared with those without UV treatment. Electrofusion between UV-irradiated protoplasts of Satsuma mandarin (donor) with those of Jincheng (C. sinensis Osbeck, recipient), a local cultivar of superior quality, gave rise to regeneration of several lines of shoots, which failed to root despite enormous endeavors. Ploidy analysis via flow cytometry and chromosome counting showed that four selected shoots were either diploid, triploid or tetraploid. Random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) confirmed the shoots, irrespective of their ploidy level, as putative somatic hybrids. Cleaved amplified polymorphism sequences (CAPS) demonstrated that the shoots predominantly got their cytoplasmic components, in terms of chloroplast (cp) and mitochondrion DNA, from Jincheng, along with possible recombination of cpDNA in some shoot lines. The current data indicated that UV-based asymmetric fusion could also be employed in citrus somatic hybridization with the intention of creating novel germplasms, which may provide an alternative approach for cultivar improvement.