Sugar beet (Beta vulgaris L.) haploid parthenogenesis in vitro: factors and diagnostic characters

Traditional obtaining of inbred lines and hybrids in sugar beet breeding requires a long time and is labour-consuming because of 2-year cycle of plant development, self- and cross-incompat-ibility, and inbreeding depression. To induce genotypic diversity in initial population, biotechnology methods including haploid parthenogenesis are promising. We have shown that, when inducing sugar beet ( Beta vulgaris L.) haploids in vitro, express-diagnostics using phenotypic and embryological characters that are representative of periods of flowering shoots, organs and buds development, and stages of embryo sac, ovule and pollen grains formation is effective. The regenerative activity is observed in ovules of bud 1 to bud 25 located on ear part of pleochasium upward from the open flower. The nuclei and cells of female gametophyte of isolated ovules under in vitro conditions are capable of neoplasm at all stages of development, but the 8-nuclear or 7-celled embryo sacs are the most appropriate to morphogenesis and switching of development program from gametophyte to sporophyte type. Critical period of embryo sac development has been beforehand determined from the accompanying embryological characters - the presence of single-nuclear and two-three-celled pollen grains of anthers located in the same bud as ovules. The results we obtained indicate that hormonal composition of the Gamborg's B-5 (B5) medium is an important factor that effectively regulates direction of morphogenetic development in isolated ovules through direct regeneration (embryoidogenesis) or via callus (hemorhizogenesis) that is the evidence of totipotency of both sexual and somatic cells in the explant. The obtained data on in vitro reproduction of haploid regenerants add to available scientific notion of morphogenetic potential specificity in sugar beet plants. Stabilizing selection used to produce double haploids promotes detection of valuable morphological features of the regenerants. Determination of chromosome and chloroplast numbers in stomata guard cells as well as isozyme electrophoretic mobility (for Adh-1, Mdh-1, Mdh-2, Me-1, Idh-1, Idh-2, Gdh-1 loci) can serve as markers when inducing haploidy and producing homozygous restitution lines of sugar beet. Efficiency of RFLP-analysis method using Hind III restrictase that has allowed for the first time to identify haploid microclones according cytoplasm type is shown. Molecular markers have indicated that regenerants with normal cytoplasm (N) have one PCR-product of 800 bp in length not digested by Hind III. Two fragments (320 bp and 480 bp) of 800 bp product digestion are found in cytoplasmic male sterile (CMS) forms (S) that reflects combination of recessive and dominant genes. Obtaining haploid regenerants with sterile cytoplasm from initial population is of great theoretical and practical importance for sugar beet breeding thus facilitating the problem of producing homozygous lines with CMS and high-productive hybrids on the sterile basis.