Optimization in Agrobacterium-mediated transformation of Anthurium andraeanum using GFP as a reporter

Abstract

Although Agrobacterium-mediated transformation protocols for many economically important plant species have been well established, protocol for a number of flowering plants including Anthurium andraeanum remains challenging. In this study, we report success in generating transgenic Anthurium andraeanum cv Arizona using Agrobacterium GV3101 strain harboring a binary vector carrying gfp as a reporter gene. The possibility of facilitating the screening process for transgenic plants expressing functional proteins using gfp marker was explored. In order to realize high transformation efficiency, different explant sources including undifferentiated callus pieces and petioles were compared for their regeneration efficiency and susceptibility to Agrobacterium-mediated transformation. We also optimized the concentration of AS added to co-cultivation media. Genomic PCR revealed that 11 of the 22 resistant plantlets regenerated on selective medium were successfully transformed. Green fluorescence was observed using a fluorescence microscope in 7 of the 11 PCR-positive plants, indicating GFP was expressed stably in the transformed Anthurium andraeanum. The highest transformation efficiency obtained in this study was 1.71% (percentage of explants with transgenic shoots in total explants) when callus explants were used as starting material and 125 μmol l -1 AS was added during the co-cultivation process.

Genetic transformation and molecular research in Anthurium: progress and prospects

“…Petiole explants from A. andraeanum 'Arizone' did not form callus when transformed with A. tumefaciens strain GV3101 if the level of AS was lower than 75 mg/l (Zhao et al 2010) but the level of recovery reached 0.64 % when callus were transformed even if the cocultivation medium was free of AS. In the experiments of Hosein et al (2012), the type and developmental stage of explants also had a very strong effect on transient transformation efficiency.…”

“…Despite successful transformation and the presence of the uidA gene being confirmed in the genome by Southern blot hybridization, GUS protein and activity were not detected in uidA transformed lines (Chen and Kuehnle 1996). The gene for green fluorescent protein (GFP) was more readily expressed (Hayden and Christopher 2004;Zhang et al 2009;Zhao et al 2010) and this reporter gene is more suitable and reliable for promoter studies in Anthurium. CaMV35S and senescence-activated promoters from Arabidopsis thaliana (sag12) and Anthurium (anth17) were compared in transient assays.…”

“…Transformation of anthurium has been carried out mainly by using Agrobacterium-mediated transformation (Kuehnle and Chen 1994;Chen and Kuehnle 1996;Chen et al 1997;Kuehnle et al 2001Kuehnle et al , 2004aKhaithong et al 2006Khaithong et al , 2007Zhou et al 2008;Zhao et al 2010;Fitch et al 2011;Hosein et al 2012) with only two studies employing biolistic methods (Hayden and Christopher 2004;Matsumoto et al 2013) and one study employing a laser microbeam puncture method (Zhang et al 2009) (Table 1). Kuehnle et al (2001) genetically transformed anthurium by dipping internodes into a suspension of A. tumefaciens under weak light to induce callus.…”

“…When acetosyringone (AS) concentration was 100 lmol/l and infection time was 10 min, the survival rate of resistant callus and the differentiation rate of resistant buds were highest (48.65 and 11.46 %, respectively). Zhao et al (2010) successfully generated transgenic A. andraeanum 'Arizona' using A. tumefaciens GV3101 strain harboring a binary vector carrying the gfp reporter gene. The highest transformation efficiency obtained in that study was 1.71 % when callus explants were used as the API API arrowhead proteinase inhibitor, att attacin, CBF 1 C-repeat binding factor 1, gfp green fluorescent protein gene, uidA b-glucuronidase gene, hptII hygromycin phosphotransferase II gene, nptII neomycin phosphotransferase II gene, NOS nopaline synthase promoter, OcIdeltaD86 modified rice cysteine protease inhibitor, PR1 pathogenesis-related protein 1, Shiva-1 cecropin-like lytic peptide Plant Cell Tiss Organ Cult An H2 medium is mentioned but no information about it could be found in the paper Plant Cell Tiss Organ Cult starting material and 125 lmol/l AS was added during cocultivation.…”

Standardization of Agrobacterium mediated genetic transformation in Indica rice cv BPT-5204

“…In rice, many factors are influencing the efficiency of T-DNA delivery to plant cell such as type of explants, optical density of Agrobacterium, infection time, co-cultivation medium with acetosyringone for vir gene induction. An excessive number of bacteria can stress plant cells and affect their regeneration potential, whereas low concentration can reduce the frequency of T-DNA transfer (Fitch et al, 2011;Zhao et al, 2010). In general, cocultivation time of 2-3 days is standard for most transformation systems (Mourgues et al, 1996;Cervera et al, 1998).…”