Variability of organ-specific gene expression in transgenic tobacco plants

Abstract

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from an Agrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene of Parasponia andersonii in front of the gus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.

A risk assessment study of plant genetic transformation usingAgrobacterium and implications for analysis of transgenic plants

“…Various antibiotics have been employed to eliminate Agrobacterium following transformation, but very few authors actually test to ensure that the antibiotics succeed. Several examples of the use of carbenicillin at 500 mg 1-1 (Radke et al, 1988;Moloney et al, 1989;da Camara Machado et al, 1992;Eapen and George, 1994) and more recently cefotaxime at 200-500 mg 1-1 Hassan et al, 1993;Jacq et al, 1993;Calfalonieri, 1994;Howe et al, 1994;van der Hoeven et al, 1994) exist, but not one tested to ensure that bacterial elimination occurred.…”

Genomic stability and stability of expression in genetically modified plants

“…Only plants displaying the desired level of expression of the new gene during several generations will pass the selection procedure. It is important to bear in mind that two co-introduced genes may differ in the individual stability of their expression as well as in their sensitivity to the selection procedure (for example see Register et al 1994;Van Der Hoeven et al 1994;Schulze et al 1995). Variations in the levels of transgene expression of independent transformants have been observed (Peach & Velten 1991;Nap et a l 1993).…”

“…The use of heterologous organ-specific promoters, which should avoid variation in expression in organs, does not necessarily result in organ-specific expression. For example, the introduction of a GUS construct driven by the root-specific par (Parasponid) haemoglobin promoter in tobacco showed that expression of the gene was also found in the leaves as well as in the roots (Van der Hoeven et al 1994). Even the use of promoters from highly conserved proteins does not allow constitutive expression in the same organ.…”

A plant scaffold attached region detected close to a T-DNA integration site is active in mammalian cells

“…So far, the expression level of genes introduced into the plant genome is not predictable and can vary substantially between individual plants transfonmed with the same gene construct (1)(2)(3)(4), in some cases to the extent of loss of organ specificity (5). Gene expression has been positively as well as negatively correlated with gene copy numbers (2,6,7).…”

“…This promoter has been shown to confer root specific expression in transgenic tobacco (22), however, leakiness of organ specificity has been observed in a number of transformants of tobacco (5,22) and petunia (Dietz and Landsmann, unpublished). The leakiness in organ specificity of the PAR promoter in some transformants may be ascribed to influences of the integration locus.…”