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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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The development of putative transgenic sorghum plants. a The initiation stage—immature embryo-derived calli ready for microprojectile transformation; b putative transgenic shoots on selective (30 mg/L G418) regeneration medium at 6 weeks; c putative transgenic plantlet on selective (30 mg/L G418) rooting medium at 4 weeks; d putative transgenic plants in containment glasshouse
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Fig6: The development of putative transgenic sorghum plants. a The initiation stage—immature embryo-derived calli ready for microprojectile transformation; b putative transgenic shoots on selective (30 mg/L G418) regeneration medium at 6 weeks; c putative transgenic plantlet on selective (30 mg/L G418) rooting medium at 4 weeks; d putative transgenic plants in containment glasshouse

Mentions: IEs at 9–11 days old after initiation were used for microprojectile transformation. Six IEs were placed at the center of a shallow petri dish 15 × 90 mm containing osmotic medium and stored for 2–3 h in the dark prior to bombardment (Fig. 6a). Operation of the particle inflow gun (PIG) was conducted as described by Vain et al. (1993) (Grootboom et al. 2010). For plasmids, DNA delivery occurred via 0.6 μm gold particles (0.42 mg per shot). The distance from the filter holder to the target cells was adjusted to 18.5 cm and the helium pressure was modified to 1,000 kPa. As much as 5 μg of each pUKN plasmid and pGEM-ubi-gfp plasmid were mixed and then equally loaded into the receptacle for six shots. A 500-μm nylon mesh screen was placed 8 cm above the target tissue and a vacuum of approximately −90 kPa was generated prior to shooting with a time duration of 0.05 s. Post-bombardment, IEs were kept on osmotic medium for 3–4 h before being transferred onto CIM.


Highly efficient sorghum transformation.

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

The development of putative transgenic sorghum plants. a The initiation stage—immature embryo-derived calli ready for microprojectile transformation; b putative transgenic shoots on selective (30 mg/L G418) regeneration medium at 6 weeks; c putative transgenic plantlet on selective (30 mg/L G418) rooting medium at 4 weeks; d putative transgenic plants in containment glasshouse
© Copyright Policy
Related In: Results  -  Collection

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

Fig6: The development of putative transgenic sorghum plants. a The initiation stage—immature embryo-derived calli ready for microprojectile transformation; b putative transgenic shoots on selective (30 mg/L G418) regeneration medium at 6 weeks; c putative transgenic plantlet on selective (30 mg/L G418) rooting medium at 4 weeks; d putative transgenic plants in containment glasshouse
Mentions: IEs at 9–11 days old after initiation were used for microprojectile transformation. Six IEs were placed at the center of a shallow petri dish 15 × 90 mm containing osmotic medium and stored for 2–3 h in the dark prior to bombardment (Fig. 6a). Operation of the particle inflow gun (PIG) was conducted as described by Vain et al. (1993) (Grootboom et al. 2010). For plasmids, DNA delivery occurred via 0.6 μm gold particles (0.42 mg per shot). The distance from the filter holder to the target cells was adjusted to 18.5 cm and the helium pressure was modified to 1,000 kPa. As much as 5 μg of each pUKN plasmid and pGEM-ubi-gfp plasmid were mixed and then equally loaded into the receptacle for six shots. A 500-μm nylon mesh screen was placed 8 cm above the target tissue and a vacuum of approximately −90 kPa was generated prior to shooting with a time duration of 0.05 s. Post-bombardment, IEs were kept on osmotic medium for 3–4 h before being transferred onto CIM.

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