The colchicine is an alkaloid that stops cellular division provoking the chromosome set doubling (one of the cell daughters retain both chromosome set). Chromosome duplication or polyploidy is associated with an enlargement of organs (flowers and leaves), an intensification of colours, hardier and more robust plants, thicker and more rigid foliage, an apparent increase in the tolerance to different stresses, and the resistance to diseases and pests. For this reason polyploidization is recognized as a source of evolution and domestication of flowering plants, this property is profited for the plant breeding, especially in ornamental crops.

The genus Scoparia is native from Argentina. This genus shows an interesting diversity in shapes and colours of the flowers, as well as different growing habits (Table 1).

However, flower size represents a problem in terms of their potential commercial value because in all of five Scoparia species they are too small.

The purpose of the present study is to explore the Scoparia species germplasm by means of in vitro polyploidization in order to improve their ornamental qualities. In the present study we describe the in vitro micropropagation protocol for four species (five different accessions) of Scoparia spp, as well as the development of a new variety of S. montevidiensis using in vitro colchicine polyploidization as a breeding tool.

Individuals of S. montevidiensis var. montevidiensis, S. montevidiensis var. glandulifera, S. nudicaulis, S. hasleriana and S. dulcis were collected from the North-Eastern Argentine provinces and maintained under greenhouse conditions.

In order to obtain an adequate number of individuals that allow making colchicine experiments, S. montevidiensis var. glandulifera was used as a model in a preliminary assay to test the multiplication rate of nodal segments. The culture was made in glass culture tubes containing complete MS solidified medium supplemented with 0.0; 0.25; 0.5 and 1.00 mg/L naphtalen acetic acid (NAA) and 6, Bencyl amino purine (BAP), both plant growth regulators (in all possible combinations), 20 g/l sucrose, 7,0 g/L agar, pH = 5,7. The physical culture conditions were 16:8 light-darkness and 22ºC temperature.

Table 2 shows that the best response was obtained with treatment containing 0.25 mg/l BAP without NAA. Figure 1 shows the tissue culture progress of S. montevidiensis var. glandulifera.

This treatment and the same physical conditions were used to test the in vitro behaviour of nodal segments (length: 0.5 cm) of the other five Scoparia accessions. Table 3 shows that except for S. hasleriana with a performance of little callus proliferation, followed by browning of the callus and ending with explants death, the other accessions showed a good behaviour under the culture conditions proposed and no differences between them were found.

As mentioned before, colchicine provokes an incresase of nuclear DNA amount, which can be verified using a flow citometer. This device is based on the measure of the fluorescence emitted by fluoresceine stained nucleus when light is full upon them. As the colorant used is DNA specific, the measured fluorescence is proportional to the DNA amount.

For the polyploidization assay 180 nodal segments were distributed in 10 different colchicine treatments and their controls (see Table 3). From this experiment 379 plants were recovered and analyzed, from those 15 were chimeric plants (plant with cells containing different amounts of DNA) and 4 plants were solid tetraploids (all cells of the plant with the DNA amount doubled). The flow citometer graphics are shown in Figure 2.

Significant differences were observed for the size of flowers, leaves, and stem diameter among the tetraploid plants and between them and the control. (see Table 4 and Figure 3).

The present study is the first report of the application of this biotechnological methodology in Scoparia genus. Tissue culture combined with the polyploidization treatment showed to be a very interesting alternative to obtain the needed variability in Scoparia genus to start a breeding program.