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

Abstract

Dendrobium orchid is one of the most popular cut flower and potted plants. In this study, a protocol for efficient genetic transformation of D. nobile-type orchids was established by co-cultivating 21 day-old protocorms for 3 days with Agrobacterium tumefaciens strain EHA101 carrying pIG121Hm harboring β-glucuronidase (GUS) gene as reporter gene and hygromycin phosphotransferase (hpt) gene as selectable marker gene. After selection of the infected protocorms on New Dogashima (ND) medium containing 10 g l −1 maltose, 30 mg l −1 hygromycin and 20 mg l −1 meropenem for 3 months followed by the culture on hygromycin-free recovery medium for 1 month, secondary protocorm-like bodies (PLBs) produced on this medium were again transferred onto secondary selection (regeneration) medium. Plantlets were successfully regenerated from these secondary PLBs after the transfer. The highest transformation efficiency of 27.3% was obtained when protocomrs were inoculated with 10 times diluted Agrobacterium solution (OD 600 =0.1) for 300 min. Transformation of the selected plants was confirmed by GUS assay, PCR and Southern blot analysis. This protocol could be adopted to produce transgenic D. nobile-type orchids with various traits such as novel flower color and resistances to biotic and abiotic stresses.

A perspective on orchid seed and protocorm development

“…The ability for protocorms or protocorm sections to generate PLBs, make them ideal candidates for transformation studies. Successes are now reported regularly in the literature using protocorms and PLBs as explants (Phlaetita et al 2015 ). One can expect rapid progress in this area of research in the near future.…”

Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale

“…The EHA105 A. tumifaciens strain was provided by Takara Biomedical Technology and the pCambia1301-35SN is kindly provided by Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences. The transformation protocol used for the Dendrobium orchid was developed by previous study (Men et al, 2003; da Silva et al, 2011; Hsing et al, 2016; Phlaetita et al, 2015; da Silva et al, 2016). …”

“…We focused on two: overexpression of exogenous genes in the host and silencing of endogenous genes of the host. To achieve overexpression, many reports have published methods for introducing exogenous genes into various Orchidaceae species by bombardment or Agrobacterium -mediated transformation (Yu et al, 2001; Mishiba et al, 2005; Lu et al, 2007; Zhang et al, 2010; da Silva et al, 2011, 2016; Hsing et al, 2016; Phlaetita et al, 2015). To achieve gene silencing, just a few reports showed RNAi-mediated gene knock-down in Dendrobium Sonia (Liu et al, 2014) and Oncidium hybrid orchid (Liu et al, 2014).…”

The effects of β-lactam antibiotics and hygromycin B on de novo shoot organogenesis in apple cv. Golden Delicious

“…An optimum choice of a selective agent can make the process of selection of transformed cells more efficient. Hygromycin B, an aminoglycoside antibiotic produced by Streptomyces hygroscopicus [23] has been frequently used in the selection process in a large number of plant species [24][25][26]. It effectively inhibits the translocation of mRNA and tRNAs on the ribosome in both bacteria and eukaryotes, and has a subtle effect on decoding fidelity [27,28].…”