Agrobacterium-mediated transformation of protocorm-like bodies in Cattleya

Abstract

Protocorm-like bodies (PLBs) of Cattleya orchid CM2450 were cocultivated with Agrobacterium tumefaciens strain EHA101 carrying either plasmid pIG121-Hm harboring genes coding for neomycin phosphotransferase II, hygromycin phosphotransferase, and b-glucuronidase (GUS) or plasmid pBBRacdS harboring these same genes along with a gene coding for 1-aminocyclopropane-1-carboxylate (ACC) deaminase. PLBs were maintained in a liquid New Dogashima (ND) medium and then added to a bacterial suspension culture (OD 600 & 0.6) yielding dilution ratios of either 1:10 or 1:40, and these were incubated for either 30 min, 3 h, or 6 h. Hygromycinresistant secondary PLBs were induced after 4 weeks of culture on an ND medium containing 1.0 mg L -1 naphthaleneacetic acid (NAA), 0.1 mg L -1 benzyladenine (BA), 10 mg L -1 hygromycin, 20 mg L -1 meropenem, 10 g L -1 sucrose, and solidified with 2.5 g L -1 gellan gum. The highest frequency of transformation was obtained when PLBs were inoculated with 1:10 Agrobacterium liquid culture for 3 h. The transformation of hygromycin-resistant plantlets regenerated from different sites of inoculated PLBs was confirmed by histochemical GUS assay, polymerase chain reaction (PCR) analysis, and Southern blot hybridization.

High efficiency <i>Agrobacterium</i>-mediated transformation of <i>Dendrobium</i> orchid using protocorms as a target material

“…Although usefulness of high bacterium concentration has been reported as an imported factor for achieving transformation of orchids Mishiba et al 2005;Shrestha et al 2007;Zhang et al 2010), it has less effect in the present study, i.e. 1 : 10 dilution (OD 600 ≈0.1) gave slightly higher transformation efficiency than 1 : 50 dilution only at 300 min inoculation time and no differences was found at 30 min inoculation (Table 1).…”

“…In the last 15 years, genetic transformation of orchids has been reported using Agrobacterium-mediated method by targeting protocorm-like bodies (PLBs) as material for Phalaenopsis (Belarmino and Mii 2000;Chai et al 2002;Sjahril and Mii 2006), Cymbidium (Chen et al 2002;Chin et al 2007), Vanda (Shrestha et al 2007), Cattleya (Zhang et al 2010), Oncidium (Liau et al 2003) and Dendrobium (Men et al 2003b;Nan et al 1998;Yu et al 2001). It is natural to use PLBs as the target for genetic transformation because most cultivars of these commercially important orchids are vegetatively propagated by tissue culture, and hence targeting the cultivars with superior traits is usually needed.…”

Agrobacterium-mediated genetic transformation of Cattleya with an Odontoglossum ringspot virus replicase gene sequence

“…In our previous study, we established a transformation procedure in Cattleya using PLB as the explant material (Zhang et al 2010). In the present study, therefore, we report successful result on the production of transgenic plants of Cattleya with ORSV replicase gene sequence, which was introduced into PLBs by developing an improved protocol of Agrobacterium-mediated transformation system.…”

Agrobacterium-mediated transformation of Dendrobium orchid with the flavanone 3-hydroxylase gene

“…Chai et al (2002) and Liau et al (2003) reported screening of Phalaenopsis and Oncidium using hygromycin at a concentration of 3 and 5 mg/L, respectively. In addition, hygromycin at a concentration of 10 mg/L has also been used to screen transgenic Vanda and Cattleya plants (Shrestha et al, 2010;Zhang et al, 2010).…”