Oncidium and Odontoglosum orchid species have reduced display lives and are thus commercially less important than Phalaenopsis. One approach to prolonging display life permanently is to transform Oncidium and Odontoglossum with the ethylene receptor mutant gene etr1-1 from Arabidopsis under control of a flower specific promoter; this should reduce their sensitivity to exogenous ethylene. To achieve this it will be necessary to establish an efficient regeneration protocol using somatic embryogenesis and a routine Agrobacterium tumefaciens-mediated transformation procedure. Protocorm-like bodies (PLBs) of both orchid genera were regenerated from leaf tip explants. Leaf tips and PLBs, cultured in liquid and solid media, were compared as targets for genetic transformation. No transgenic shoots were obtained from leaf tips, while PLBs of Oncidium and Odontoglossum cultured on solid medium were successfully transformed with an expression vector containing nptII and gus genes driven by the cauliflower mosaic virus (CaMV) 35S promoter. Applying the A. tumefaciens strain EHA 105, transformation efficiencies of 1.3-2.7% were achieved for the investigated genotypes. Transformation with etr1-1 gene was achieved subsequently. Oncidium 'Sweet Sugar' has been successfully transformed and validated by PCR and Southern analysis.

Flowers of the complex orchid hybrid Burrageara 'Stefan Isler Lava Flow' had been shown previously to react sensitively to ethylene. Via Agrobacterium tumefaciens, the mutant ethylene receptor ETHYLENE RESPONSE 1 (etr1-1) from Arabidopsis thaliana under the control of the flower-specific promoter FLOWER BINDING PROTEIN 1 (fbp1) from Petunia hybrida was transferred to Burrageara. One single-copy event was analyzed in this study aiming to investigate the expression of the fbp1::etr1-1 transgene in different plant and flower organs by quantitative RT-PCR and the reaction of flowers and inflorescences to ethylene. It was shown that the heterologous promoter led to high expression levels in the perianth of the orchid flowers compared to low levels in leaves and roots. The expression shift to the first whorl (sepals) described here corresponds to extended expression of endogenous B class MADS box homeotic genes in orchids in general. The transgenic plants grew and developed similar to the wild-type plants, except for slightly faster rooting in vitro and smaller flowers. Flower longevity was improved by 7 days in 10 ppm ethylene. Moreover, bud drop starting at day 5 of incubation of inflorescences in 10 ppm ethylene in the wild-type was efficiently prevented for at least 19 days in the fbp1::etr1-1 transgenic plants. The function of the tissue-specific promoter fbp1 and the mutant receptor etr1-1 was shown for the first time in a monocotyledonous plant.

Oncidium, eine Gattung der Orchidaceae, weist ein weites Spektrum an verschiedenen Blütenfarben und -formen auf und konnte daher an wirtschaftlicher Bedeutung gewinnen. Die traditionelle Züchtung ist bei dieser Gattung sehr zeitaufwändig, weshalb es Ziel dieser Untersuchung war, ein effizientes Protokoll zur In-vitro-Regeneration über somatische Embryogenese und zum Agrobacterium tumefaciens-vermittelten Gentransfer zu entwickeln, damit der Züchtungsprozess verkürzt werden kann. Ausgehend von In-vitro-Sprossen der Sorte 'Sweet Sugar', die über Sprossspitzenkultur etabliert worden waren, wurde ein verlässliches Protokoll zur Regeneration von protocorm like bodies (PLBs) etabliert. Dabei stellten sich Blattspitzen als überlegene Explantatart heraus, bei denen nach zwölf Wochen eine Regenerationsrate von 60 % beobachtet werden konnte. Eine effiziente Regeneration konnte auf 1/2 MS (Murashige und Skoog, 1962) Medium mit Thidiazuron und in vollständiger Dunkelheit erzielt werden. Mit diesem Protokoll zur Regeneration wurde die Dauer der Vorkultur der Blattspitzen für den Agrobacterium tumefaciensvermittelten Gentransfer variiert. In histochemischen GUSTests nach vier und sechs Wochen wurden Transformationsereignisse an Blattspitzenexplantaten nachgewiesen. Es regenerierten die ersten PLBs aus diesen Versuchen. Die molekulargenetischen Untersuchungen zum Beweis für eine erfolgreiche Transformation sind in Arbeit.

No abstract

No abstract