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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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Southern blot hybridization of 14 putative transgenic lines with nptII probe. Lines from left to right: M DNA ladder. N NC, P equivalent single copy control (pUKN plasmid), 1–14 individual transgenic lines
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Fig5: Southern blot hybridization of 14 putative transgenic lines with nptII probe. Lines from left to right: M DNA ladder. N NC, P equivalent single copy control (pUKN plasmid), 1–14 individual transgenic lines

Mentions: PCR results confirmed the presence of the NPTII gene in all transgenic lines and no nptII escapes were observed (Fig. 4a). The result of gfp PCR analysis showed that 18 of 25 plants were positive (Fig. 4b). Overall, the co-transformation frequency of the gfp and nptII genes was 72%. Southern hybridisation confirmed the integration of the nptII gene into the genome of all T0 plants (Fig. 5). Single copy and multiple copies of nptII gene were found in various plant lines. Among the 25 transgenic lines, 24% had a single nptII gene copy, 28% had two to three copies and 48% had at least four copies.Fig. 4


Highly efficient sorghum transformation.

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

Southern blot hybridization of 14 putative transgenic lines with nptII probe. Lines from left to right: M DNA ladder. N NC, P equivalent single copy control (pUKN plasmid), 1–14 individual transgenic lines
© Copyright Policy
Related In: Results  -  Collection

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

Fig5: Southern blot hybridization of 14 putative transgenic lines with nptII probe. Lines from left to right: M DNA ladder. N NC, P equivalent single copy control (pUKN plasmid), 1–14 individual transgenic lines
Mentions: PCR results confirmed the presence of the NPTII gene in all transgenic lines and no nptII escapes were observed (Fig. 4a). The result of gfp PCR analysis showed that 18 of 25 plants were positive (Fig. 4b). Overall, the co-transformation frequency of the gfp and nptII genes was 72%. Southern hybridisation confirmed the integration of the nptII gene into the genome of all T0 plants (Fig. 5). Single copy and multiple copies of nptII gene were found in various plant lines. Among the 25 transgenic lines, 24% had a single nptII gene copy, 28% had two to three copies and 48% had at least four copies.Fig. 4

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