Agrobacterium-mediated transformation of Phalaenopsis by targeting protocorms at an early stage after germination

Abstract

A transformation procedure for phalaenopsis orchid established by using immature protocorms for Agrobacterium infection was aimed at the introduction of target genes into individuals with divergent genetic backgrounds. Protocorms obtained after 21 days of culture on liquid New Dogashima medium were inoculated with Agrobacterium strain EHA101(pIG121Hm) harboring both beta-glucuronidase (GUS) and hygromycin resistance genes. Subculture of the protocorms on acetosyringone-containing medium 2 days before Agrobacterium inoculation gave the highest transformation efficiencies (1.3-1.9%) based on the frequency of hygromycin-resistant plants produced. Surviving protocorms obtained 2 months after Agrobacterium infection on selection medium containing 20 mg l(-1) hygromycin were cut transversely into two pieces before transferring to recovery medium without hygromycin. Protocorm-like bodies (PLBs) proliferated from pieces of protocorms during a 1-month culture on recovery medium followed by transfer to selection medium. Hygromycin-resistant phalaenopsis plants that regenerated after the re-selection culture of PLBs showed histochemical blue staining due to GUS. Transgene integration of the hygromycin-resistant plants was confirmed by Southern blot analysis. A total of 88 transgenic plants, each derived from an independent protocorm, was obtained from ca. 12,500 mature seeds 6 months after infection with Agrobacterium. Due to the convenient protocol for Agrobacterium infection and rapid production of transgenic plants, the present procedure could be utilized to assess expression of transgenes under different genetic backgrounds, and for the molecular breeding of phalaenopsis.

Agrobacterium-Mediated Transformation of Indonesian Orchids for Micropropagation

“…A reproducible methodology for the genetic transformation of orchids, and better recognition of the factors affecting the transformation process, are needed in order to support this objective. Previous studies have reported orchid transformation either directly through the delivery of marker genes such as those encoding Escherichia coli β-glucuronidase (GUS) and Aequorea victoria green fluorescent protein (GFP) into plant cells by particle bombardment (Anzai et al, 1996), or indirectly through the use of A. tumefaciens (Belarmino and Mii, 2000;Chia et al, 1994;Mishiba et al, 2005;Chan et al, 2005;. Recently, we developed a convenient method for genetic modification of Phalaenopsis amabilis orchids using A. tumefaciens (Semiarti et al, 2007) in which intact protocorms (young orchid seedlings) were used for transformation.…”

Induction of Somatic Embryogenesis through Overexpression of ATRKD4 Genes in Phalaenopsis “Sogo Vivien”

“…it can easily and quickly produced in large quantities as it comes from the growth of seeds, it has a high level of regeneration, and it allows transfer of genes into protocorm (Mishiba et al, 2005).…”

“…This compound is highly reactive towards biomolecules, which cause damage of plant tissue followed by browning and necrosis (Kuta and Tripathi, 2005). Several studies of genetic transformation using Agrobacterium with transformation targets in the form of protocorm-like bodies (plb) and orchid protocorm obtained transformation effi ciency of 4.3% in Cattleya (Zhang et al, 2010); 1.91% in Phalaenopsis hybrid (Mishiba et al, 2005); 1.7% in Phalaenopsis amabilis (Semiarti et al, 2007); and 1.3 to 2.7% in Oncidium and Odontoglossum (Raffeiner et al, 2009). To improve the transformation effi ciency, there are many studies using a variety of treatments, such as the provision of antioxidants, desiccation of transformation target before and after infection with Agrobacterium, length of co-cultivation time, density of Agrobacterium tumefaciens used in the transformation, temperature of co-cultivation process, and the addition of surfactant at the time of Agrobacterium tumefaciens infection to the explants (Opabode, 2006).…”

“…The medium used for preculture contained 2,4-D, tomato extract and acetosyringone, which aimed to keep the meristematic level of protocorm, protected from oxidative damage, and to improve the response level of protocorm when gene transfer was performed (Mishiba et al, 2005 ;Semiarti et al, 2010;Dwiyani et al, 2010). In this research, co-cultivation was carried out in 3 days to optimize the integration of T-DNA into the genome of protocorms.…”

“…In this research, co-cultivation was carried out in 3 days to optimize the integration of T-DNA into the genome of protocorms. For the elimination of bacteria, ah ah antibiotics meropenem was used as it could effectively disturbed the growth of Agrobacterium (Mishiba et al, 2005). The selection of transformant candidates was done in 6 weeks with intensive subcultured for every 2 weeks on selection medium containing hygromycin 10 mg.L -1 .…”