Agrobacterium tumefaciens‐mediated sorghum transformation using a mannose selection system

Abstract

SummaryA dual-marker plasmid containing the selectable marker gene, manA , and the reporter gene, sgfp , was used to transform immature sorghum embryos by employing an Agrobacteriummediated system. Both genes were under the control of the ubi1 promoter in a binary vector pPZP201. The Escherichia coli phosphomannose isomerase (PMI) gene, pmi , was used as the selectable marker gene and mannose was used as the selective agent. The sgfp gene encoding green fluorescence protein (GFP) was the reporter gene and served as a visual screening marker. A total of 167 transgenic plants were obtained from nine different embryogenic callus lines grown on a selection medium containing 1% − 2% mannose. Embryoids and shoots regenerated via embryogenesis, that showed strong GFP fluorescence, were selected from two sorghum genotypes: C401, an inbred line, and Pioneer 8505, a commercial hybrid.The GFP accumulation in transgenic plants was observed with a dissecting stereomicroscope.The integration and expression of the manA gene was confirmed by Southern blot and Western blot analyses, and the feasibility of manA selection was demonstrated by the chlorophenol red (CPR) assay. Our results indicated that transgenes segregated in the Mendelian fashion in the T 1 generation. The conversion of mannose to a metabolizable fructose carbon source is beneficial to plants. In addition, except in soybean and a few legumes, no endogenous PMI activity has been detected in plant species, indicating that PMI is useful in the transformation of sorghum. In addition, PMI has no sequence homology to known allergens. Optimization of this selection system for sorghum transformation provides an efficient way to produce transgenic plants without using antibiotic or herbicidal agents as selectable markers, and our results showed that the transformation efficiency reached 2.88% for Pioneer 8505 and 3.30% for C401, both values higher than in previously published reports.

The impact of over-expression of NPK1 gene on growth and yield of sorghum under drought stress

“…Further screening was conducted through PCR analysis, the transformation frequency was calculated and found to be within the cited range (Emani et al, 2002;Gao et al, 2005b;Zhao et al, 2000;Howe et al, 2006) from 0.2 to 4.5%, even though these reports were on highly regenerable and transformable genotypes, e.g. P898012 and TX430.…”

“…To sustain callus regenerability, bialaphos concentration was not increased beyond 1.5 mg/L. T h i s i s because non-transgenic plantlets may escape the bar selection system (Gao et al, 2005b).…”

Sorghum Transformation: Overview and Utility

“…In addition the positive selectable marker gene phosphomannose isomerase (PMI) (Joersbo and Okkels 1996) has been shown to be a rather robust selection system for the identification of transgenic plants (Negrotto et al 2000), including sorghum (Gao et al 2005b). …”

Genetic transformation of Sorghum bicolor

“…This research group from Kansas State University later attempted to enhance resistance to fungal diseases and drought tolerance through tlp gene from rice (Thaumatin-like protein). Along with the visual reporter gene gfp, the tlp was introduced into explants (immature zygotic embryos) of three sorghum genotypes (two inbreds: Tx 430 and C401; and a commercial hybrid, Pioneer 8505) via Agrobacterium-mediated transformation method (Gao et al, 2005b) and eventually generated transgenic sorghum plants.…”