Agrobacterium tumefaciens-mediated transformation of poinsettia, Euphorbia pulcherrima, with virus-derived hairpin RNA constructs confers resistance to Poinsettia mosaic virus.
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.
Clarke
Jihong Liu
JL
Plant Health and Plant Protection Division, Norwegian Institute for Agricultural and Environmental Research, Hoegskoleveien 7, 1432 Aas, Norway. jihong.liu-clarke@bioforsk.no
Spetz
Carl
C
Haugslien
Sissel
S
Xing
Shaochen
S
Dees
Merete W
MW
Moe
Roar
R
Blystad
Dag-Ragnar
DR
D016960:Agrobacterium tumefaciens / Q000235:genetics; D028481:Euphorbia / Q000196:embryology / Q000235:genetics* / Q000821:virology; D018014:Gene Transfer Techniques; D010935:Plant Diseases / Q000821:virology*; D030821:Plants, Genetically Modified / Q000196:embryology / Q000235:genetics* / Q000821:virology; D034622:RNA Interference; D016337:RNA, Catalytic / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes; D046830:Tymoviridae / Q000201:enzymology / Q000472:pathogenicity*
D016337:RNA, Catalytic; C418481:hairpin ribozyme