Expression Variability of Marker Genes during Plant Regeneration from Protoplasts of R1 Transgenic Mustard (Brassica juncea) Via Somatic Embryogenesis

Abstract

Summary Expression of marker genes, neomycin phosphotransferase (NPT) II and s-glucuronidase (GUS), in the R1 progeny originating from a transgenic mustard plant line was investigated. The R1 shoots grown in the absence of kanamycin for 6 months showed reduced GUS activity in various tissues and an inability to form roots in the presence of kanamycin. Protoplasts isolated from these shoots expressed GUS at a frequency of 1-5% after 3, 10, 14 and 28d of culture, whereas wild type protoplasts did not show GUS activity throughout the process of somatic embryogenesis. Exogenous application of 20 μM 5-aza-cytidine greatly enhanced the frequency of GUS expressing cells, ranging from 26 to 34%, but not for the wild type during somatic embryogenesis. In protoplast-derived plants, GUS activity was detected in 4 out of 9 plants analysed. Southern analysis revealed that the NPTII gene was present in all GUS positive plants, but absent in GUS negative plants. The pattern of GUS expression in leaves was age-dependent irrespective of the plant developmental stage. GUS was strongly expressed in mature leaves, which was concomitant with an accumulation of a 1.8 kb GUS transcript. In contrast, GUS activity was either absent or occurred only at serrated tips of the young leaves, in which the GUS transcript levels were barely detectable. This study shows transgene instability in R1 mustard plants, which may be due to DNA methylation and/or deletion of the transgenes.