Alfalfa belongs to the most important forages not only in Slovakia, but also in the whole world. This is confirmed also by the fact that alfalfa is grown on 32 million hectares of arable land. From the aspect of its importance and because of intensive selection and development of local highly productive populations, genetic variability of this crop has declined radically in the last decades. Therefore development of new alfalfa varieties by conventional breeding methods is still more and more difficult. In vitro techniques provide an important contribution to the breeding programmes, where classical breeding is insufficient or it fails. But the main assumption of using in vitro techniques for the breeding purposes is regeneration of whole plants from differentiated cells (BINGHAM et al., 1975). In our work, we aimed at the screening of high regeneration ability genotypes in one of the last released Slovak alfalfa varieties Lucia and development of breeding material with high regeneration ability by means of repeated cycles of somatic embryogenesis induction. Moreover, obtained progenies of regenerants were evaluated according to the morphological and agronomical traits. Alfalfa regenerants obtained using in vitro culture were analysed cytologically. Our effort was to analyse somatic number of chromosomes of alfalfa regenerants and to verify in this way phenomenon of somaclonal variability occurring in alfalfa cell cultures. In the first cycle of screening alfalfa (Medicago sativa L.) genotypes of Lucia variety for regeneration ability, 13 regenerants of R₀ generation have been obtained (KRAIC et al., 1994). In 1996, representative part of the seed S₁ and OP₁ was used for the screening of both progenies of regenerants for in vitro regeneration ability (second screening cycle) with the aim to obtain alfalfa population with a higher regeneration ability of genotypes than original variety. At the same time some economic characteristics of clonal as well as S₁ and OP₁ generations of regenerants were tested in field conditions. During the whole cycle of screening for ability to produce somatic embryos the explants were evaluated for the frequency of callus formation, callogenesis intensity, frequency of embryogenic callus production, and frequency of embryogenic genotypes, which were used as the main criteria for selection of embryogenic genotypes. In the field crops, colour of flower, health conditions, average plant height, average number of stems/plant, and average plant weight after each cutting were studied. Results of field experiments have shown a high uniformity of the clones in flower colour. All of the plants were similar to the parental forms and as for the studied morphological traits, no somaclonal variants were found out. Comparison of S₁ and OP₁ populations and clonal progenies under the field conditions pointed out genotype differences in the number of stems/plant, in the average plant weight, as well as average plant height after ascertaining the average of the data obtained in the first harvest year after three cuttings. Inbreeding depression of the studied traits was revealed in the progeny obtained by selfpollination. From the results it follows that changes in chromosome number in alfalfa regenerants ranged from 8 to 28 % of the whole number of analyzed metaphases. The lowest occurrence of metaphases with variance in chromosome number was revealed in regenerant R5, namely 8 %. Up to 20 % of changes in chromosome number were assessed in regenerants R4, R7 (13 %), and R1, R8 (19 %). Other regenerants (besides regenerants that have not been analyzed, R2, R3, R9) had occurrence of aneuploid and polyploid cells in the progeny higher than 20 %. The highest value was recorded in the regenerant R6 (28 %). The results obtained confirm that there exists the possibility of selecting alfalfa population from commercial variety Lucia enriched with the trait of in vitro high regeneration ability.

Reference

Bingham, E.T., Hurley, L.V., Kaatz, D.M., Saunders, J.W. (1975): Crop Sci., 15: 719-721.

Kraic, J., Už’k, M., Hauptvogel, P. (1994): Biologia (Bratislava), 49: 59-63.