Utilization of the bar gene to develop an efficient method for detection of the pollen-mediated gene flow in Chinese cabbage (Brassica rapa spp. pekinensis)

Abstract

To develop an efficient screening method for detection of the transgene in Chinese cabbage (Brassica rapa spp. pekinensis) utilizing Basta spray, optimal conditions for Basta application were examined in this study. Two transgenic Chinese cabbage lines were obtained through Agrobacterium-mediated transformation and used as transgenic positive controls in the Basta screening experiment. Differential concentrations of glufosinate-ammonium were sprayed into three different growth stages of 12 commercial Chinese cabbage cultivars. The results showed that no plants could survive higher than 0.05% glufosinateammonium, and plants at the 2-3 leaf stage were most vulnerable to glufosinate-ammonium. On the other hand, no damage was observed in the transgenic control plants. Reliability of the Basta spray method was proven by showing perfect co-segregation of the tolerance to glufosinate-ammonium and the presence of the bar gene in T 1 segregating populations of the transgenic lines, as revealed by both PCR and Southern blot analyses. Using the developed Basta screening method, we tried to investigate the transgene flow through pollen dispersal, but failed to detect any transgene-containing non-transgenic Chinese cabbages whose parents had been planted adjacent to transgenic Chinese cabbages in field conditions. However, the transgene was successfully detected using Basta spray from the non-transgenic plants bearing the transgene introduced by hand-pollination. Since the Basta spray method developed in this study is easy to apply and economical, it will be a valuable tool for understanding the mechanism of gene flow through pollen transfer and for establishing a biosafety test protocol for genetically modified (GM) Chinese cabbage cultivars.

Application of the acetolactate synthase gene as a cisgenic selectable marker for Agrobacterium-mediated transformation in Chinese cabbage (Brassica rapa ssp. pekinensis)

“…The recalcitrance of Chinese cabbage to transformation could be due to the low efficiency of Agrobacterium infection, low regeneration efficiency of the infected tissues, and low selection efficiency for transformed cells (Lee et al 2004;Takasaki et al 1997). Bacterial antibiotic resistance genes such as nptII and hpt (Cho et al 2001;Jun et al 1995;Kuginuki et al 2001;Lee et al 2004;Park et al 2005;Shin et al 2003;Vanjildorj et al 2009;Zhang et al 2000), herbicide resistance genes such as bar (Lim et al 2007), and a mannose-6-phosphate isomerase gene from Escherichia coli (Min et al 2007) have been used as selectable markers for Chinese cabbage transformation. However, to date, no plant-derived gene has been used as a selectable marker for Chinese cabbage transformation.…”