Sterile poplars prevent outcrossing in forest ecosystems

Topic

Genetically modified trees will be available for commercial use in a few years. They are likely to be used mainly in plantations.

The safety assessment of genetically modified woody plants must take into account a possible spread of the transgenes through cross-pollination and seed dispersal. In order to ensure that transgenes do not spread into natural forest tree populations, sterility concepts are being developed for woody plants.

Since there are no conventional strategies for producing and incrossing natural sterility, genetically engineered male and/or female sterility is an important approach.

The aim of this project was to develop a genetic engineering approach – production of male and female sterility using various gene constructs – using the aspen as a model. The research focussed in particular on the following questions:

• Are the sterility mechanisms already tested in herbaceous plants also capable of functioning in aspens?

• Can viable transgenic sterile lines be selected for a release experiment in the greenhouse?

• Can stable expression of the sterility genes be guaranteed in the long term?

Summary

Within this project it was possible to produce transgenic early-flowering aspens and to equip them with various sterility gene constructs. Female plants equipped with sterility genes were transferred to the greenhouse as early as 2002. Male-sterile lines followed in the spring of 2003. Several of these lines have flowered in the laboratory and have been assessed with regard to the expression of the sterility constructs. One of the main aims of the project has therefore already been achieved, although more detailed investigations into the gene activity of the sterility genes in poplars are still to be carried out.

Alternative methods for promoting flowering, e.g. treating poplars with hormones or growth inhibitors, have so far failed to show any stimulating effect on flowering. These plants will, however, continue to be tested with regard to their flowering ability.

Experiment description

Unlike annual herbaceous plants, tree species take several years to flower for the first time. In order to shorten the time to flowering, the project used transgenic aspen lines that flower after a period of only a few months to three years. Poplars normally flower only after eight years.

Transgenic early-flowering aspen lines

The research project started by using existing transgenic aspen lines bearing the rolC gene (root locus C gene), which have been the subject of much research in the greenhouse and in the field. The rolC gene comes from the soil bacterium Agrobacterium rhizogenes and causes phenotypic changes in the transgenic plants. Dwarf growth and early flower formation are typical traits of these transgenic plants.

In addition, the project also produced genetically modified aspen lines bearing an early flowering gene from the birch (BpMADS4), the Leafy gene from Arabidopsis, the rolD gene from Agrobacterium rhizogenes or the XX gene (name changed) from Arabidopsis .

Approaches that involve treating the poplars with various growth regulators or growth inhibitors to induce early flowering were also tested.

Genetically engineered introduction of sterility genes

Various strategies for producing sterility were tested by transferring gene constructs with various sterility genes into transgenic early-flowering plants and into non-transgenic control plants. The constructs contained tissue-specific control elements (promoters ). The promoters used for this purpose caused sterility-inducing gene expression only in certain plant parts, e.g. in the pollen, in the stigma or in the pollen sacs (tapetum) where pollen formation takes place.

The genetically modified plants were to be transferred to the greenhouse immediately after transformation in order to allow any early flower formation to be investigated. The plan was to cultivate early flowering transgenic lines in the laboratory as well.

Results

Male flower on an early flowering Leafy transgenic poplar line, in vitro Female flower on an early flowering Leafy transgenic poplar line, in vitro Male single flowers on an early flowering XX transgenic poplar line, in vitro Female catkin on an early flowering rolC transgenic poplar line in the greenhouse Male catkin on a non-transgenic poplar line

Transgenic early flowering aspen lines Transformations with the early flowering constructs (BpMADS4, Leafy, rolD, XX) were successfully carried out and the transgenic plant lines were transferred to the greenhouse. Promotion of the flowering ability, which had originally been planned with the rolC gene, was significantly improved using the Leafy and XX genes. The Leafy and XX transgenic poplars flowered after only two to twelve months, thereby demonstrating the best stimulation of flowering compared with the usual three to four years for rolC transgenic poplar lines and the eight years or more for “normal” poplars. Genetically engineered introduction of sterility genes So far, this project has transferred sterility genes into normally flowering and early flowering Leafy transgenic plants. The transfer of sterility genes into normally flowering poplars was successful. Female plants with the sterility genes BpMADS1::Barnase were transferred to the greenhouse as early as 2002. Building on the results to date, early flowering male-sterile lines (with the 35S::Leafy construct) were produced which have already been tested using Southern analysis. Several of these lines have flowered in vitro and have been tested for expression of the sterility constructs. However, the Leafy transgenic poplars produced only single flowers (no catkins), showed disturbed growth and clone-dependent fertility. It therefore made sense to look for alterative ways of inducing early flower formation. New experiments have shown that using the XX gene from Arabidopsis in poplars enables much better promotion of flower formation. XX transgenic poplars flower just as early as Leafy transgenic poplars, but have the advantage of forming complete catkins instead of single flowers. The project began transformations using this XX gene in December 2004. The XX gene is under the control of either the 35S promoter from the cauliflower mosaic virus or a heat-inducible promoter.