A b s t r a c tTraditional 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-incompatibility, 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.Keywords: sugar beet (Beta vulgaris L.) haploid ...

Here we consider aspects of the application of biotechnological methods to rapid creation, propagation, and maintenance of plants with improved or new traits in sugar beet breeding. The results of the works carried out in these fields by the Federal State Budgetary Scientific Institution “The A.L. Mazlumov All-Russia Research Institute of Sugar Beet” are reviewed. A close association between morphological and physiological changes in in vitro cultured organs and tissues, on the one hand, and breeding traits, on the other hand, which allows the development of experimental systems for non-amphimictic plant reconstruction is shown. The influence of in vitro growth conditions on haploid cells of unfertilized sugar beet ovules in the course of obtaining doubled haploid lines with high degree of homozygosity and maintenance of valuable breeding properties is considered. As compared to common inbreeding, this method shortens the time for development of homozygous material from 10–12 to 3–5 years, which is of great importance for speeding-up the breeding process. The results of studies on the culturing of mature sugar beet zygotic embryos based on in vitro selective systems have made it possible to improve the adaptive potential of plants and to provide complex resistance to environmental stress factors. Strict selection under abiotic stress conditions allowed creation of sugar beet isogenic lines with tolerance of drought, salinity, and soil acidity. It is shown that the proposed original design of mass-scale microclonal in vitro reproduction and deposition of elite plants as components of highly productive hybrids can be used to obtain seeds of uniform high-quality breeding material. The technologies developed by biotechnological methods are a topical and innovative direction of inquiry, since the application of these techniques to sugar beet breeding will promote obtaining of competitive hybrids with a set of commercially valuable traits. The combination of biotechnology methods, including tissue culture, and traditional breeding techniques is expected to provide an opportunity to obtain a new starting material to develop domestic varieties and hybrids of new generation with heterosis effect and a wide resistance spectrum persisting across generations.

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High efficiency of the cultivation of unfertilized sugar beet ovules and preparation of haploid regenerants (microclones) of pollinators – maintainers of О-type sterility and MS forms of the RMS 120 hybrid components has been shown. A technological method that accelerates the creation of new uniform starting material is proposed. It speeds up the breeding process two to threefold. The identification of haploid regenerants with sterile cytoplasm in initial populations is of great theoretical and practical importance for breeding, as it facilitates the production of homozygous lines with cytoplasmic male sterility and high-performance hybrids on sterile basis. As shown by molecular analysis, a single-nucleotide polymorphism never reported hitherto is present in the mitochondrial genome of the haploid plant regenerants. It allows identification of microclones as fertile and sterile forms. It has been found that DNA markers of the sugar beet mitochondrial genome belonging to the TR minisatellite family (TR1 and TR3) enable reliable enough identification of haploid microclonal plants as MSor O-type forms. Fragments of 1000 bp in length have been detected in monogenic forms in the analysis of 11 sugar beet plants cultured in vitro by PCR with the OP-S4 random RAPD primer. Testing of the OP-S4 marker’s being in the same linkage group as the genes responsible for expression of the economically valuable trait monogermity demonstrates its relative reliability. By the proposed method, dihaploid lines (DH) of the male-sterile form and the О-type sterility maintainer of the RMS 120 sugar beet hybrid have been obtained in in vitro culture. These lines are highly uniform in biomorphological traits, as proven under field conditions.

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