Optimization of Agrobacterium-mediated transformation parameters for Melastomataceae spp. using green fluorescent protein (GFP) as a reporter

Yong, Wilson Thau Lym; Abdullah, Janna Ong; Mahmood, Maziah

doi:10.1016/j.scienta.2006.03.005

Cited by 25 publications

(17 citation statements)

References 11 publications

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“…Therefore, a reliable transformation and regeneration system with a low degree of chimerism and high regeneration frequency is a prerequisite (May et al 1995;Sági et al 1995). There are several factors that affect the transformation efficiency, such as Agrobacterium density, infection time, co-cultivation duration and conditions (Yong et al 2006;Vrbová et al 2013), and temperature (Li et al 2003). To date, there is no report of Agrobacterium-mediated or direct gene transformation of medicinal ginger, Boesenbergia rotunda.…”

Section: Introductionmentioning

confidence: 99%

Stable integration of mgfp5 transgenes following Agrobacterium-mediated transformation in Boesenbergia rotunda cell suspension culture

Wong

Zoolkefli

Karim

et al. 2015

Frontiers in Life Science

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Boesenbergia rotunda, a herb in the ginger family, contains numerous beneficial compounds, such as flavonoids, flavones and cyclohexenyl chalcone derivatives, that have great potential for pharmaceutical applications. However, the low concentration of the bioactive compounds limits their commercial application. In this study, a simple and reliable Agrobacterium-mediated transformation protocol for B. rotunda cell suspension culture was successfully developed. The minimal inhibitory concentration and natural tolerance of the selective agent, hygromycin, against the cells were 20 mg l −l and 30 mg l −l in liquid media and solid media, respectively. The highest number of transformed regenerants (18 ± 0.00 per ml settled cell volume) was recorded when cells were infected with Agrobacterium tumefaciens harbouring pCAMBIA1304 for 10 min and co-cultivated for 2 days. Prolonged infection time ( > 10 min) and co-cultivation period ( > 2 days), however, did not increase the transformation efficiencies. The results clearly show that infection and co-cultivation periods strongly influenced the transformation efficiency in ginger. The transformed cells were recovered and showed green fluorescent signals under ultraviolet excitation. An intense blue colour was observed in the transformed cells after β-glucuronidase (GUS) histochemical staining, further confirming the functionality of the GUS enzymes in the regenerants. Polymerase chain reaction analysis of 3-, 6-, 9-and 12-month-old transformed cells confirmed that the protocol enabled stable integration of the mgfp5 gene. Moreover, the comparatively high number of transformed regenerants in this study made it possible to generate a large number of transgenic cells in a short period, which would be useful for high-throughput functional screening of enzymes involved in the biosynthetic pathways of bioactive compounds.

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Section: Introductionmentioning

confidence: 99%

Stable integration of mgfp5 transgenes following Agrobacterium-mediated transformation in Boesenbergia rotunda cell suspension culture

Wong

Zoolkefli

Karim

et al. 2015

Frontiers in Life Science

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“…According to Zuraida et al (2011), delivery of gusA gene was most efficient in rice cultivar MR232 by using Agrobacterium cell density of OD 600nm 0.6. Yong et al (2006) reported that the highest rate of genetic transformation of members of the Melastomataceae family with Agrobacterium LBA4404 was obtained at a cell density of 0.8 OD 600nm (1x10 7 cfu ml -1 ). They also found that higher concentrations decreased transformation efficiency for shoot and node explants.…”

Section: Resultsmentioning

confidence: 99%

Optimization of various factors affecting Agrobacterium-mediated transformation and regeneration of putative transgenic Malaysian MR219 rice with Bacillus SP 289 endo-β-1,3-1,4-glucanase gene

et al. 2017

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Sheath blight disease caused by Rhizoctonia solani is the most destructive diseases in rice cultivation. Development of transgenic indica rice MR219 through Agrobacterium tumefaciens strain EHA 105 harbouring the plasmid pCAMBIA 1305.2 with endo-beta-1,3-1,4-glucanase gene from Bacillus SP 289 is one of the strategies to engineer disease resistance. Four optimisation parameters were examined such as Agrobacterium culture cell density (0.1 to 1.0 based on OD600nm), callus immersion time in the Agrobacterium culture (30 to 120 minutes), duration of the subsequent drying time (15 to 120 minutes) and co-cultivation period (1 to 6 days). Hygromycin-resistant embryogenic calli developed after 8 to 10 weeks of transformation. Improved transformation rates were achieved when calli were incubated with an Agrobacterium suspension with a culture density of OD 600nm 0.2 for 30 mins, followed by 90 mins of drying time and co-cultivation for 3 days. PCR analysis of the transformants confirmed the presence of Bacillus SP 289 endo-beta-1,3-1,4-glucanase and hpt genes in the rice genome. The transgenic rice plants obtained in this study will be tested against sheath blight disease.

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“…Green fluorescent protein (GFP) is a very useful and reliable marker for screening putative transformants of various crops or ornamental plants including florist's chrysanthemum (Zvereva and Romanov, 2000;RakosyTican et al, 2007;Yong et al, 2006). Here, the ability of the RbcS promoter to support a much stronger expression of the GFP gene compared to the commonly used CaMV35S promoter was shown.…”

Section: Gfp Expression Levelmentioning

confidence: 99%

A novel expression cassette for the efficient visual selection of transformed tissues in florists chrysanthemum (Chrysanthemum morifolium Ramat.)

Mao¹,

Stoopen²,

Jongsma³

et al. 2011

Afr. J. Biotechnol.

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Constructs carrying visual reporter genes coupled with efficient promoters could facilitate the process of identification and selection of stable transformants in recalcitrant crops. Here, a novel construct utilizing a ribulose-1,5-bisphosphate carboxylase (RbcS) promoter combined with the green fluorescent protein (GFP) reporter gene to initiate very high expression of GFP in florist's chrysanthemum (Chrysanthemum morifolium Ramat.) was described. Based on this expression cassette, a new regeneration protocol using leaf discs as explants was developed for the Agrobacterium-mediated transformation of Chrysanthemum genotype '1581', and a transformation efficiency of 7% was obtained. The expression of two different GFP constructs targeted to either cytosol or plastids was compared in transgenic lines. Both GFP constructs were expressed at such a high level that the green fluorescence dominated red fluorescence in the leaf tissues, allowing easy observation and microdissection of transformed tissues even without a GFP filter. Under normal light, plants with GFP targeted to plastids had a light green phenotype deriving from the high GFP expression. Quantitative reverse transcriptional PCR analysis showed that the plastid targeted construct with intron had significantly higher steady state transcript levels of GFP mRNA. This novel expression cassette may allow direct visual selection of transformed tissues independent of antibiotic selection in a wide range of plant species.

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Optimization of Agrobacterium-mediated transformation parameters for Melastomataceae spp. using green fluorescent protein (GFP) as a reporter

Cited by 25 publications

References 11 publications

Stable integration of mgfp5 transgenes following Agrobacterium-mediated transformation in Boesenbergia rotunda cell suspension culture

Stable integration of mgfp5 transgenes following Agrobacterium-mediated transformation in Boesenbergia rotunda cell suspension culture

Optimization of various factors affecting Agrobacterium-mediated transformation and regeneration of putative transgenic Malaysian MR219 rice with Bacillus SP 289 endo-β-1,3-1,4-glucanase gene

A novel expression cassette for the efficient visual selection of transformed tissues in florists chrysanthemum (Chrysanthemum morifolium Ramat.)

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