Oct 30, 2002

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Research into gene expression in transgenic sugar beet/mangold hybrids

(1996 – 1998) Federal Biological Research Centre for Agriculture and Forestry (BBA), Institute for Plant Virology, Microbiology and Biosafety; Braunschweig

Topic

Because of their close biological relationship, transgenic sugar beet can cross-pollinate with the mangold. The purpose of this research project was to assess the possible consequences of such a gene transfer.

Hybrids of transgenic virus-resistant sugar beet and mangold were produced using precise hand-pollination. These plants and their progeny were examined to investigate the following questions:

  • Does the gene expression change over the course of the plants’ development or does it remain stable under different location and climate conditions?
  • How does the expression of the coat protein (viral protein) behave under rizomania infection and infection-free conditions?

In addition to the gene for the coat protein of the rizomania virus, which confers resistance to this virus, the transgenic sugar beet with which the mangold hybrids had been produced also contained a marker gene (nptII) and a gene for herbicide resistance (bar gene).

Summary

  • While the expression of the herbicide resistance gene remained stable throughout, the expression of the virus resistance gene under the control of the 35S promoter appears to depend heavily on insolation and temperature. On hot days with many hours of sunshine, an almost complete silencing of the virus resistance gene was observed in about half of the transgenic plants.
  • It was not possible to identify the cause of the disabling.
  • No conclusive answer was found concerning to what extent the level of virus resistance is dependant on the gene expression.

Experiment description

Plants from field experiments of another research project were examined:

  • transgenic virus-resistant sugar beet/mangold hybrids on a rizomania infection site (Mainz) and on an infection-free site (Aachen),
  • mangold plants and non-transgenic sugar beet/mangold hybrids as a control,
  • at the infection site, also non-transgenic sugar beet of a variety susceptible to the rizomania virus.

Bolters were removed regularly during the vegetation period to rule out pollen transfer.

To assess the effect of different development stages and climate conditions on the expression of the coat protein gene, leaf and root samples were taken at regular intervals.

The expression of the coat protein gene and the rizomania infection of the plants from the infection site were detected using ELISA. The gene expression of the herbicide resistance gene was assessed using enzyme tests. PCR was used to test for the presence of the transgenes in the hybrids.

Results

Gene expression over the course of the plant’s development and under different site and climate conditions

  • In both release years (1995 and 1996) expression of the herbicide resistance gene was found to be stable for all transgenic hybrids at all sampling times.
  • Because of metrological problems (with ELISA), it was possible to measure only very small amounts of the rizomania coat protein in 1995. It was therefore almost impossible to differentiate between expressing and non-expressing plants.
  • The tendency for the expression to be higher at lower temperatures and with less insolation was confirmed in 1996 with higher values. The expression of the coat protein gene was heavily dependent on the time the sample was taken.

Expression under infection and infection-free conditions

  • It was not possible to determine conclusively whether there is a correlation between gene expression and the level of virus resistance, since the virus infection on the trial fields was uneven. Even most of the virus-susceptible control plants were not infected. Under these conditions, a reduced infection rate was observed among the transgenic virus-resistant plants.

Isolated incidences (approx. one in 28) were found of transgenic plants with a strong virus infection. In these the virus protection did not work, presumably because the coat protein gene was disabled and the coat protein could therefore not be formed.

DNA methylation (chemical modification) is frequently the cause of gene disabling. The relevant transgenic plants were therefore examined more closely in the area of the coat protein gene. However, no methylation was found in the examined DNA area. The lack of expression of the coat protein is therefore probably due to other causes.