Abstract
Background Phalaenopsis orchid (Phal. orchid) is visually attractive and it is important economic floriculture species. Phal. orchids have many unique biological features. However, investigation of these features and validation on their biological functions are limited due to the lack of an efficient transformation method.ResultsWe developed a heritable and efficient Agrobacterium- mediated transformation using protocorms derived from tetraploid or diploid Phal. orchids. A T-DNA vector construct containing eGFP driven by ubiquitin promoter was subjected to transformation. An approximate 1.2–5.2 % transformation rate was achieved. Genomic PCR confirmed that hygromycin selection marker, HptII gene and target gene eGFP were integrated into the orchid genome. Southern blotting indicated a low T-DNA insertion number in the orchid genome of the transformants. Western blot confirmed the expression of eGFP protein in the transgenic orchids. Furthermore, the GFP signal was detected in the transgenic orchids under microscopy. After backcrossing the pollinia of the transgenic plants to four different Phal. orchid varieties, the BC1 progenies showed hygromycin resistance and all surviving BC1 seedlings were HptII positive in PCR and expressed GFP protein as shown by western blot.ConclusionsThis study demonstrated a stable transformation system was generated for Phal. orchids. This useful transformation protocol enables functional genomics studies and molecular breeding.Electronic supplementary materialThe online version of this article (doi:10.1186/s40529-016-0146-6) contains supplementary material, which is available to authorized users.
Highlights
Phalaenopsis orchid (Phal. orchid) is visually attractive and it is important economic floriculture species
We developed an alternative transformation procedure using protocorms generated by germination of seeds derived from diploid Phal. aphrodite and tetraploid Phal. cultivars
Total protein was isolated from the leaf of orchid and 30 μg total protein was loaded into 10 % acrylamide gel, electrophoresed, and transferred onto polyvinylidene fluoride (PVDF) membrane for antibody probing
Summary
Phalaenopsis orchid (Phal. orchid) is visually attractive and it is important economic floriculture species. Orchids have very unique biological features such as crassulacean acid metabolism (CAM) photosynthesis that uptake CO2 at night (Guo and Lee 2006), epiphytic habitat with high water and nutrient usage efficiency, unique flower pattern formation (Su et al 2013b), symbiosis with mycorrhizae (Yumiko et al 2007), immature embryos (seeds without endosperm) in mature capsules (Yu and Goh 2001). To elucidate these interesting and novel biological features, validation of gene function using stable transformation. Protocorm transformations have been proposed to be simpler than subculture of PLBs (Mishiba et al 2005; Semiarti et al 2007)
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