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Highly efficient sorghum transformation.

Liu G, Godwin ID - Plant Cell Rep. (2012)

Bottom Line: Transgenic events were confirmed by both PCR screening and Southern hybridization of genomic DNA from primary transgenics (T₀).Co-transformation rate of the nptII and gfp genes was 72% in these experiments.The segregation of nptII and gfp in T₁ progenies was observed utilizing fluorescence microscopy and geneticin selection of seedlings indicating both were inherited in the T₁ generation.

View Article: PubMed Central - PubMed

Affiliation: School of Agriculture and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.

ABSTRACT
A highly efficient microprojectile transformation system for sorghum (Sorghum bicolor L.) has been developed by using immature embryos (IEs) of inbred line Tx430. Co-bombardment was performed with the neomycin phosphotransferase II (nptII) gene and the green fluorescent protein (gfp) gene, both under the control of the maize ubiquitin1 (ubi1) promoter. After optimization of both tissue culture media and parameters of microprojectile transformation, 25 independent transgenic events were obtained from 121 bombarded IEs. The average transformation frequency (the total number of independent transgenic events divided by the total number of bombarded IEs) was 20.7% in three independent experiments. Transgenic events were confirmed by both PCR screening and Southern hybridization of genomic DNA from primary transgenics (T₀). More than 90% of transformants were fertile and displayed normal morphology in a containment glasshouse. Co-transformation rate of the nptII and gfp genes was 72% in these experiments. The segregation of nptII and gfp in T₁ progenies was observed utilizing fluorescence microscopy and geneticin selection of seedlings indicating both were inherited in the T₁ generation. The transformation procedure, from initiating IEs to planting putative transgenic plantlets in the glasshouse, was completed within 11-16 weeks, and was approximately threefold more efficient than the previously reported best sorghum transformation system.

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GFP expression under the blue light at the wavelength of 395 nm of the OLYMPUS BX60 fluorescence microscope; a Tx430 immature embryo-derived callus 3 days after bombardment; b root tip of non-transgenic Tx430 plantlet; c root tip of transgenic Tx430 plantlet; d 3-day-old T1 seedling
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Fig3: GFP expression under the blue light at the wavelength of 395 nm of the OLYMPUS BX60 fluorescence microscope; a Tx430 immature embryo-derived callus 3 days after bombardment; b root tip of non-transgenic Tx430 plantlet; c root tip of transgenic Tx430 plantlet; d 3-day-old T1 seedling

Mentions: Strong gfp gene expression was observed in all bombarded IEs with at least 20 GFP foci. More than 100 GFP foci were spotted in about 80% of IEs (Figs. 2, 3a). When examining root tips of putative transgenic plantlets, the gfp gene expression was shown to be systemically green under the fluorescent microscope (Fig. 3c). No GFP was found in root tips of non-transgenic plantlets and the root tips of these plantlets displayed yellow color under the fluorescent microscope (Fig. 3b).Fig. 2


Highly efficient sorghum transformation.

Liu G, Godwin ID - Plant Cell Rep. (2012)

GFP expression under the blue light at the wavelength of 395 nm of the OLYMPUS BX60 fluorescence microscope; a Tx430 immature embryo-derived callus 3 days after bombardment; b root tip of non-transgenic Tx430 plantlet; c root tip of transgenic Tx430 plantlet; d 3-day-old T1 seedling
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351618&req=5

Fig3: GFP expression under the blue light at the wavelength of 395 nm of the OLYMPUS BX60 fluorescence microscope; a Tx430 immature embryo-derived callus 3 days after bombardment; b root tip of non-transgenic Tx430 plantlet; c root tip of transgenic Tx430 plantlet; d 3-day-old T1 seedling
Mentions: Strong gfp gene expression was observed in all bombarded IEs with at least 20 GFP foci. More than 100 GFP foci were spotted in about 80% of IEs (Figs. 2, 3a). When examining root tips of putative transgenic plantlets, the gfp gene expression was shown to be systemically green under the fluorescent microscope (Fig. 3c). No GFP was found in root tips of non-transgenic plantlets and the root tips of these plantlets displayed yellow color under the fluorescent microscope (Fig. 3b).Fig. 2

Bottom Line: Transgenic events were confirmed by both PCR screening and Southern hybridization of genomic DNA from primary transgenics (T₀).Co-transformation rate of the nptII and gfp genes was 72% in these experiments.The segregation of nptII and gfp in T₁ progenies was observed utilizing fluorescence microscopy and geneticin selection of seedlings indicating both were inherited in the T₁ generation.

View Article: PubMed Central - PubMed

Affiliation: School of Agriculture and Food Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.

ABSTRACT
A highly efficient microprojectile transformation system for sorghum (Sorghum bicolor L.) has been developed by using immature embryos (IEs) of inbred line Tx430. Co-bombardment was performed with the neomycin phosphotransferase II (nptII) gene and the green fluorescent protein (gfp) gene, both under the control of the maize ubiquitin1 (ubi1) promoter. After optimization of both tissue culture media and parameters of microprojectile transformation, 25 independent transgenic events were obtained from 121 bombarded IEs. The average transformation frequency (the total number of independent transgenic events divided by the total number of bombarded IEs) was 20.7% in three independent experiments. Transgenic events were confirmed by both PCR screening and Southern hybridization of genomic DNA from primary transgenics (T₀). More than 90% of transformants were fertile and displayed normal morphology in a containment glasshouse. Co-transformation rate of the nptII and gfp genes was 72% in these experiments. The segregation of nptII and gfp in T₁ progenies was observed utilizing fluorescence microscopy and geneticin selection of seedlings indicating both were inherited in the T₁ generation. The transformation procedure, from initiating IEs to planting putative transgenic plantlets in the glasshouse, was completed within 11-16 weeks, and was approximately threefold more efficient than the previously reported best sorghum transformation system.

Show MeSH
Related in: MedlinePlus