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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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Constructs, a the pUKN construct and b the pGEM-ubi-gfp construct
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Fig1: Constructs, a the pUKN construct and b the pGEM-ubi-gfp construct

Mentions: Two plasmids, pUKN and pGEM-ubi-gfp (Dugdale et al. 1998), which contain nptII encoding NPTII and gfp encoding GFP, respectively, were kindly provided by Scott Hermann, Bureau of Sugar Experimental Stations (BSES) Limited. Both genes were driven by the maize ubiquitin1 (ubi1) promoter and terminated with A. tumefaciens nopaline synthase 3′ termination signal (nos) (Fig. 1a, b).Fig. 1


Highly efficient sorghum transformation.

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

Constructs, a the pUKN construct and b the pGEM-ubi-gfp construct
© Copyright Policy
Related In: Results  -  Collection

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

Fig1: Constructs, a the pUKN construct and b the pGEM-ubi-gfp construct
Mentions: Two plasmids, pUKN and pGEM-ubi-gfp (Dugdale et al. 1998), which contain nptII encoding NPTII and gfp encoding GFP, respectively, were kindly provided by Scott Hermann, Bureau of Sugar Experimental Stations (BSES) Limited. Both genes were driven by the maize ubiquitin1 (ubi1) promoter and terminated with A. tumefaciens nopaline synthase 3′ termination signal (nos) (Fig. 1a, b).Fig. 1

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