A chimeric green fluorescent protein gene as an embryogenic marker in transgenic cell culture of Nicotiana plumbaginifolia Viv.

Abstract

In order to check the aptitude of the green fluorescent protein (GFP) as in vivo marker of embryogenesis several variants of the GFP structural gene were expressed in Nicotiana plumbaginifolia under the transcriptional control of seed-specific unknown seed protein (USP) and legumin B4 (LeB4) gene promoters. As control the GFP genes were constitutively expressed under the control of cauliflower mosaic virus 35S promoter. Transgenic plants with a stable high level of GFP expression were selected and used to establish a direct somatic embryogenesis system derived from mesophyll protoplasts. Tissue specificity and developmental regulation of the GFP expression were investigated for all three promoter activities by Western-blotting and fluorescence microscopy. The GFP genes under the control of the USP promoter were expressed in very early stages during somatic and zygotic embryogenesis, whereas the GFP chimeric genes controlled by LeB4 promoter were expressed in late developmental stages. After the first day of in vitro protoplast culture the cells showing GFP expression controlled by the USP promoter were selected from the cells without GFP expression by fluorescence activated cell sorting. Selected cells with GFP expression were able to regenerate shoots and roots in vitro whereas selected cells without GFP expression only showed undifferentiated callus growth. Application of a stronger auxin 2,4- d as commonly used for embryogenesis induction to in vitro cultivated protoplasts resulted not only in callus growth by hindrance or interruption of the realization of morphogenetic propagation but also caused down regulation of USP promoter activity. We, therefore, conclude that the chimeric gene composed of the USP promoter and the GFP structural gene may serve as an artificial marker gene in plant embryogenesis.