Agrobacterium-mediated transformation of protocorm-like bodies in Cymbidium

Abstract

Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for beta-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L(-1) gellan gum-solidified NDM containing 10 g L(-1) sucrose, 20 mg L(-1) hygromycin and 40 mg L(-1) meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 muM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization.

Efficient production of transgenic plantls of Vanda through sonication-assisted Agrobacterium-mediated transformation of protocorm-like bodies

“…However, inoculation for 480 min was toxic to the PLBs of Vanda and caused necrosis. Positive effect of long infection period has also been reported in other orchids such as Phalaenopsis (Belarmino and Mii 2000;Mishiba et al 2005) and Cymbidium (Chin et al 2007). Therefore, it is possible that orchids generally require the prolonged infection period to ensure the tight adhesion of bacteria onto the surface of PLBs.…”

“…After 3 days of co-cultivation, the PLBs were placed for 2 weeks on the same NDM but containing 5 mg l Ϫ1 meropenem (Meropen ® ; Sumitomo Pharmaceutical Ind. Co. Ltd., Osaka, Japan), which is highly effective for eliminating Agrobacterium without serious toxic effects on plant tissues Mii 2005, 2007;Chin et al 2007). For selection, the PLBs were transferred onto 3 g l Ϫ1 gellan gum-solidified NDM containing 30 g l Ϫ1 maltose, 10 mg l Ϫ1 meropenem and 10 mg l Ϫ1 hygromycin and cultured for 8 weeks by subculturing at 2 week-intervals onto fresh medium with the same composition.…”

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

“…In contrast, the proliferation of PLBs was inhibited when sucrose concentration in the culture medium was greater than 60 g/L. This may be due to the high osmotic stress caused by a high sugar concentration (Chin et al, 2007). In Phalaenopsis orchids, the optimum level of sucrose for PLB proliferation was found to be 40 g/L (Chai et al, 2002).…”

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

“…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.…”