SUGAR BEET (Beta vulgaris L.) HAPLOID PARTHENOGENESIS in vitro: FACTORS AND DIAGNOSTIC CHARACTERS

Abstract

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 ...

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“…In addition to environmental hazard, the introduction of 0.1-0.005 % colchicine to the nutrient media for effi cient polyploidization, the exposition of explants for 48 h to this substance led to the necrosis of regenerant growth points and degeneration of up to 40 % of experimental material. In addition, according to the researchers of polyploidization in sugar beet in vitro, the colchicination of haploids and doubling the number of chromosomes takes place in an insignifi cant number of clones, up to 38 %, but requires much labor and time of transplanting [14,17].…”

“…One of the main stages of creating homozygous lines in vitro is polyploidization and stabilization of the new lines by transferring the selected plants of these lines to cultural media using a polyploidizing substance, such as colchicine [17]. But in our research, when culturing apomictic embryos and their cloning for two-three passages on a cultural medium for sugar beet according to Gamberg et al (1968) (21) formula with the addition of BAP 0.3 mg/l and gibberellin 1 mg/l, lines of dihaploids were isolated from all the investigated selection numbers of types of regenerants, either based on new sterile cytoplasm, or regenerants, based on sterile cytoplasm (Table 3) without the addition of colchicine.…”

“…The method of inducing maternal haploids under in vitro conditions (gynogenesis), using unpollinated seed embryos decreases the period of obtaining homozygous selection materials to 1-2 years, due to genetic homogeneity of lines of the resulting dihaploids [16,17]. Zhuzhzhalova et al (2016) found that an efficient way to stimulate the yield of haploids (from 3.0 to 9.8 %) was obtained using the liquid basal nutrient medium, based on micro-and macrosalts according to Gamberg et al, to which cytokinin BAP (1.0 mg/l) was added [17].…”

Evaluating the pollen proofing of nonwoven synthetic fabric pollination control tents for sugar beet

“…Of special interest are seed-related traits since seed on plants in the open control resulted from cross pollination and that on plants in tents from pollen contamination or by parthenogenesis without contribution of pollen that could pass through the cover (Zhuzhzhalova et al, 2016). Of the six seed related traits the full analysis (a) showed significant variation between treatments for 1000-seed weight and 10-day germination (%) only (Table 2).…”

Methods of creating homozygous lines as breeding genotypes in sugar beet (Beta vulgaris) with apozygotic method of seed reproduction

“…Жужжалова й ін. (2016) дослідили, що серед найбільш ефективних технологій стимуляцію розвитку гаплоїдів від 3,0% до 9,8% забезпечувало рідке живильне середовище з включенням цитокініну БАП 1,0 мг/л [18].…”