Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus

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.

Fig. 1

Schematic representation of PnMV genome (a) and hairpin (hp) RNA constructs used in the current study (b). The locations of the primers used to generate the constructs as well as for screening purposes are presented over the PnMV genome. Sequences of the primers are presented in Table 2. The location of the probe used to detect siRNA is presented below the PnMV genome (see “Materials and methods”). CP coat protein region, R2 and R3 RNA-dependent RNA-polymerase (RdRp) regions of the PnMV genome

Fig. 2

Somatic embryogenesis in poinsettia cv. Millenium: a embryogenic structure and globular stage somatic embryos (arrows) that appeared on the callus (bar 1 mm), b cotyledonary stage of somatic embryos (bar 1 mm), c plantlets deriving from somatic embryos on RIM medium; and d regenerated plants established in the greenhouse

Fig. 3

PCR analysis. PCR positive transformants detected with primer pairs for (a) CP, (b) R2 and (c) R3 fragments respectively. Lane A1 1 kb marker; lane A2 non-transformed plant; lanesA3–5 are independent CP-transgenic lines 11-1, 11-2 and 3-1; laneA6 plasmid control. Lane B1 1 kb marker; lane B2 non-transformed plant; lanes B3-8 are R2-transgenic lines 72-1, 72-2, 75-2, 79-1, 84-2, and 84-4; lane B9 plasmid control. Lane C1 1 kb marker; lane C2 non-transformed plant, lanes C3-11 are R3 transgenic lines 18-1, 30A, 38-1, 40A, 40B, 41-2, 41-5, 56-2 and 62-1; lane C12, plasmid control. The 500 bp band of the 100 bp marker is depicted with an arrow

Fig. 4

Southern blot analysis of selected PCR positive transformants carrying pCP, pR2 and pR3 constructs. The HindIII-digested total genomic DNA was probed with a 1.5 kb probe homologous to the region of CP, R2 and R3 fragments (Fig. 1) allowing us to analyse all the three different types of transformants (CP, R2 and R3 transformants) at the same time. Lane1 plasmid control; lanes2-4 CP transformants 11-1, 11-2 and 3-1; lanes5-6 R2 transformants 72-2, 79-1; lanes 7-9 R3 transformants 38-1, 40B, 18-1; lane10 negative control

Fig. 5

Northern blot analysis of low molecular weight (LMW) RNA to detect small interfering RNAs (siRNA) in non-inoculated transgenic plants and non-inoculated control. aLane 1 control; lanes 2–3 CP transgenic lines 11-1, 3-1; lanes 4–5 R2 transgenic lines 72-1,72-2; lane 6 R3 transformant, 18-1. Ribosomal RNA is presented in (b) to indicate presence of LMW RNA in the gel. Lane 1 control; lanes 2 and 3 PCP transformed plants; lanes 4 and 5 PR2 transformed plants; lane 6 PR3 transformed plant