Development of antibiotic marker-free creeping bentgrass resistance against herbicides

Abstract

Herbicide-resistant creeping bentgrass plants (Agrostis stolonifera L.) without antibiotic-resistant markers were produced by Agrobacterium-mediated transformation. Embryogenic callus tissues were infected with Agrobacterium tumefaciens EHA105, harboring the bar and the CP4-EPSPS genes for bialaphos and glyphosate resistance. Phosphinothricin-resistant calli and plants were selected. Soil-grown plants were obtained at 14-16 weeks after transformation. Genetic transformation of the selected, regenerated plants was validated by PCR. Southern blot analysis revealed that at least one copy of the transgene was integrated into the genome of the transgenic plants. Transgene expression was confirmed by Northern blot. CP4-EPSPS protein was detected by ELISA. Transgenic plants remained green and healthy when sprayed with Basta, containing 0.5% glufosinate ammonium or glyphosate. The optimized Agrobacteriummediated transformation method resulted in an average of 9.4% transgenic plants. The results of the present study suggest that the optimized marker-free technique could be used as an effective and reliable method for routine transformation, which may facilitate the development of varieties of new antibiotic-free grass species.

Growth Performance and Field Evaluation of Herbicide-Resistant Transgenic Creeping Bentgrass

“…For efficient weed control, an expression vector to which two herbicide resistance genes were introduced was prepared and transferred into a creeping bentgrass (Lee et al, 2011). With the plant to which the genes were transferred and of which expression was confirmed (Lee et al, 2011), the effect of the herbicide treatment on the growth performance and field evaluation was investigated.…”

“…With the plant to which the genes were transferred and of which expression was confirmed (Lee et al, 2011), the effect of the herbicide treatment on the growth performance and field evaluation was investigated.…”

“…Additionally, glyphosate of 20, 80 and 320 μg/mL was treated to investigate the shikimate accumulation in the transformed creeping bentgrass plant depending on the glyphosate treatment concentrations (Lee et al, 2011Kim et al, 2008.…”

“…On the other hand, creeping bentgrass also requires ceaseless mowing in order to maintain a good green state, because it grows rapidly (Lee et al, 2011).…”

A method mediated AAVS1 recombination with Rep mRNA and homologous arms

“…Southern blot analysis was performed as described previously [22] with some modifications. Briefly, genomic DNA was harvested from each cell line.…”