Dag-Ragnar Blystad

Research Scientist

(+47) 908 72 588
dag-ragnar.blystad@nibio.no

Place
Ås H7

Visiting address
Høgskoleveien 7, 1433 Ås

Abstract

Replication of all positive-sense single-stranded RNA viruses occurs in specific structures in close association with cellular membranes. Targeting of the viral replication complex (RC) to the site of replication is mediated by the interaction of viral-encoded proteins and host factors. Electron microscope studies have shown that Poinsettia mosaic virus (PnMV, family Tymoviridae) infection is associated with the presence of vesicular structures in the chloroplasts, which indicates that the replication of PnMV might occur in association with chloroplast-derived membranes. Using computer assisted homology search, we have identified that the coat protein (CP) of PnMV shows similarity to membrane bound proteins and contains a conserved amino acid sequence motif found in members of the Alb3/Oxa1/YidC protein family. This protein family is involved in the insertion of proteins into intracellular membranes. In this study we carried out localization studies combined with confocal laser microscopy to identify the cellular localization of the PnMV CP. Transient expression of red fluorescent protein (RFP)-tagged PnMV CP in Nicotiana benthamiana protoplast was shown to localize in the chloroplast.

Abstract

Chrysanthemum stunt viroid (CSVd) was first reported in US in the 1940s and is widespread in the world wherever chrysanthemum is grown. Cryotherapy of shoot tips, a new biotechnology developed in the recent years, is a novel application of plant cryopreservation techniques that allows pathogen eradication at a high frequency. Existing studies have proven that this technique can efficiently eradicate pathogens such as virus, phytoplasma and bacterium. However, up to now, there has been no report on viroid eradication. In the present study, we attempted to establish a droplet vitrification cryotherapy method for Argyranthemum and to apply it to eradicate CSVd. Results obtained so far demonstrated that cryotherapy of shoot tips alone failed to eradicate CSVd from the infected shoot tips of Argyranthemum maderense ‘Yellow Empire’. Using in situ hybridization of CSVd and histological analysis, we found that CSVd can invade meristematic cells and at the same time, these cells were able to survive following cryotherapy. These findings explained why cryotherapy of shoot tips alone could not be efficient enough to eradicate CSVd from the diseased materials. Further studies combining cold treatment with cryotherapy are under investigation for CSVd eradication.

To document

Abstract

Euphorbia pulcherrima, poinsettia, is a non-food and non-feed vegetatively propagated ornamental plant. Appropriate plant height is one of the most important traits in poinsettia production and is commonly achieved by application of chemical growth retardants. To produce compact poinsettia plants with desirable height and reduce the utilization of growth retardants, the Arabidopsis SHORT INTERNODE (AtSHI) gene controlled by the cauliflower mosaic virus 35S promoter was introduced into poinsettia by Agrobacterium-mediated transformation. Three independent transgenic lines were produced and stable integration of transgene was verified by PCR and Southern blot analysis. Reduced plant height (21–52%) and internode lengths (31–49%) were obtained in the transgenic lines compared to control plants. This correlates positively with the AtSHI transcript levels, with the highest levels in the most dwarfed transgenic line (TL1). The indole-3-acetic acid (IAA) content appeared lower (11–31% reduction) in the transgenic lines compared to the wild type (WT) controls, with the lowest level (31% reduction) in TL1. Total internode numbers, bract numbers and bract area were significantly reduced in all transgenic lines in comparison with the WT controls. Only TL1 showed significantly lower plant diameter, total leaf area and total dry weight, whereas none of the AtSHI expressing lines showed altered timing of flower initiation, cyathia abscission or bract necrosis. This study demonstrated that introduction of the AtSHI gene into poinsettia by genetic engineering can be an effective approach in controlling plant height without negatively affecting flowering time. This can help to reduce or avoid the use of toxic growth retardants of environmental and human health concern. This is the first report that AtSHI gene was overexpressed in poinsettia and transgenic poinsettia plants with compact growth were produced.

To document

Abstract

Abstract Strict control of morphogenesis is essential in production of potted poinsettia. Commonly, this is obtained by the use of plant growth retardants (PGRs), often in combination with early morning temperature drops. Due to negative effects on human health and the environment, the use of PGRs is becoming restricted. Also, energy-saving growth regimes and periods of high temperatures limit effective use of temperature drops. In the present study the use of a high proportion of blue (B) light provided by light emitting diodes [LEDs, 20% blue (B), 80% red (R)] was compared with traditional high pressure sodium (HPS) lamps (5% B) providing similar phytochrome photostationary state to produce compact poinsettia plants. Both in the greenhouse and growth chamber, all cultivars were 20–34% shorter for LED compared to HPS grown plants. Also, leaf and bract area as well as chlorophyll content and total dry matter accumulation were lower under LED. The LED did not delay bract color formation, visible cyathia and flowering compared to HPS, and no difference in post production performance (cyathia/bract abscission or necrosis) between the two light treatments was found. The effect of end of day-red (EOD-R) lighting combination with LED and HPS supplemental lamps during the photoperiod in the greenhouse was also investigated. Reduced stem extension (13%) was observed under HPS only and for one of the two cultivars tested, whereas under the LED regime, there was no effect of EOD-R lighting.

Abstract

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.

Abstract

An infectious cDNA clone of a Norwegian isolate of Poinsettia mosaic virus (PnMV) was generated. It consisted of 6,098 nucleotides and encoded a polyprotein of 219.5 kDa. Sequence comparisons indicated that this isolate shared 98.6% (nucleotide) and 97.1% (amino acid) identity with the previously sequenced isolate from Germany. RNA transcripts derived from this cDNA were infectious in Nicotiana benthamiana. However, plants did not present typical PnMV symptoms. Furthermore, RNA transcripts from this cDNA clone were not infectious in poinsettia. Serial propagation of this cDNA clone in N. benthamiana plants restored symptom induction in this host but did not re-establish infectivity in poinsettia.