GUS expression in blueberry ( Vaccinium spp.): factors influencing Agrobacterium -mediated gene transfer efficiency

Cao, Xiaoyan; Liu, Q.; Rowland, Lisa J.; Hammerschlag, F. A.

doi:10.1007/s002990050569

Cited by 50 publications

(32 citation statements)

References 15 publications

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“…Significance was determined at p<0.05 level [20] . The percentage of GUS expression was calculated by the number of explants showing GUS positive divided by the number of explants subjected to cocultivation and multiplied by 100 [21] .…”

Section: Discussionmentioning

confidence: 99%

Agrobacterium-mediated Genetic Transformation in Cucumber (Cucumis sativus L.)

Ayyappan¹,

Selvaraj²,

Ganapathi³

et al. 2007

American J. of Biochemistry and Biotechnology

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The present study was undertaken to develop efficient transformation protocol for cucumber cv. Poinsett 76 using Agrobacterium strain EHA 105. Five-day-old mature cotyledon explants was used for transformation study. The infected explants were co-cultivated for 2 days in MS medium containing BA (1.0 mg L¯1). The selection of transformed shoots was carried out in MS medium fortified with BA (1.0 mg L¯1), Cefotaxime (300 mg L¯1) and PPT (2.0 mg L¯1). The transformed shoots were elongated in MS medium containing BA (1.0 mg L¯1), Cefotaxime (300 mg L¯1), PPT (2.0 mg L¯1) along with GA 3 (0.5 mg L¯1). The rooting of elongated shoots was achieved in MS medium with BA (1.0 mg L¯1), Cefotaxime (300 mg L¯1), PPT (2.0 mg L¯1) and IBA (0.6mg L¯1). The transient GUS expression assay and leaf disc assay were carried out in order to find transformed shoots. The molecular confirmation of transformed shoots revealed the foreign gene integration into cucumber genome.

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Section: Discussionmentioning

confidence: 99%

Agrobacterium-mediated Genetic Transformation in Cucumber (Cucumis sativus L.)

Ayyappan¹,

Selvaraj²,

Ganapathi³

et al. 2007

American J. of Biochemistry and Biotechnology

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“…Co-cultivation period is also dependent on the Agrobacterium strain used. For instance, Agrobacterium strain LBA4404 required 4 days for co-cultivation for optimal transformation in blueberry (Cao et al 1998;Cheng et al 2011).…”

Section: Effects Of Infection Times and Co-cultivation Periods On Boementioning

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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“…In similar way pre-culture also don't show any positive effect on transforming (Table 4) while Moralejo et al have studied the influence of explants pre-cultivation and reported that when seedlings were precultured for 4 -6 days, the level of GUS transient expression was significantly greater than that of control [20]. In compatible to Cervera et al and Cao et al length of co-cultivation have significant positive effect on efficiency of transformation in spinach [17,18]. By increasing co-cultivation duration number of spot per plant and percent of explants with blue spot were increased ( Table 5).…”

Section: Resultsmentioning

confidence: 79%

“…Cervera et al and Cao et al have suggested that, low efficiency of transformation in citrange explants was due to an insufficient length of co-cultivation [17,18]. However, they have also reported that it was more difficult to eliminate Agrobacterium after longer periods of co-cultivation.…”

Section: Introductionmentioning

confidence: 99%

Optimization of micropropagation and <i>Agrobacterium</i>-mediated gene transformation to spinach (<i>Spinacia oleracea</i> L.)

Naderi¹,

Zohrabi²,

Shakib³

et al. 2012

ABB

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Spinach is one of the dioecious plant which is considered as a model plant in genetic and molecular studies of sex determination because of its special characteristics such as low chromosome number and short life cycle. An efficient protocol for Spinacia oleracea Agrobacterium-mediated gene transformation was developed. The leaf disks, roots, hypocotyls and cotyledons of this plant were inoculated with LBA4404. LBA4404 carrying pCAMBIA3301 binary vector with 35SCaMV gus-int and 35SCaMV bar cassettes. Effects of two preparation condition (induction of vir genes and noninduction) were considered. Also effects of different number days of co-cultivation and pre-culture of explants were examined. After co-cultivation, the explants were transferred to regeneration medium containing 250 mg·L -1 Carbeniciline. Transient expression efficiency was calculated based on the number of blue spots per explants one week after inoculation. Based on the results of transient expression, stable transformation was carried out. After formation of callus the histochemical GUS assay was carried out on some parts of them and other parts were leaved for being regenerated.

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GUS expression in blueberry ( Vaccinium spp.): factors influencing Agrobacterium -mediated gene transfer efficiency

Cited by 50 publications

References 15 publications

Agrobacterium-mediated Genetic Transformation in Cucumber (Cucumis sativus L.)

Agrobacterium-mediated Genetic Transformation in Cucumber (Cucumis sativus L.)

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

Optimization of micropropagation and <i>Agrobacterium</i>-mediated gene transformation to spinach (<i>Spinacia oleracea</i> L.)

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GUS expression in blueberry ( Vaccinium spp.): factors influencing Agrobacterium -mediated gene transfer efficiency

Cited by 50 publications

References 15 publications

Agrobacterium-mediated Genetic Transformation in Cucumber (Cucumis sativus L.)

Agrobacterium-mediated Genetic Transformation in Cucumber (Cucumis sativus L.)

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

Optimization of micropropagation and &lt;i&gt;Agrobacterium&lt;/i&gt;-mediated gene transformation to spinach (&lt;i&gt;Spinacia oleracea&lt;/i&gt; L.)

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Optimization of micropropagation and <i>Agrobacterium</i>-mediated gene transformation to spinach (<i>Spinacia oleracea</i> L.)