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

Purpose. Investigation of cytogenetic aspects of embryological processes in the culture of immature apomictic embryos, breeding genotypes of sugar beet with cytoplasmic sterility for differentiation and selection by gametophyte reduced parthenogenesis. Methods. Cytological, biotechnological, fluorescent cytophotometry, field, laboratory. Results. The cytogenetic features of genesis of immature apomictic embryos cells induced in vitro on the 12th, 20th and 22th days of development have been investigated on the basis of CMS apozygotic lines of Beta vulgaris and alloplasmic lines of wild species Beta maritime and Beta patula. Indicators of efficiency of haploid reduced parthenogenesis in vitro in alloplasmic lines significantly exceeded the best technologies in pollen-sterile lines of sugar beet from 3.79% to 6.25% and had a value of 62.2%, 24.8%, and 16.7%, respectively. Stabilization of genome ploidy to diploid was carried out in selected breeding numbers without colchicine, based on evaluation and selection of genome ploidy using software of ploidy analyzer (AP) Partec. Conclusions. The efficiency of haploid reduced parthenogenesis induction in vitro in apozygotic CMS breeding genotypes of sugar beet as affected by genetic potential of cytoplasm and taking into account the total percentage of haploids (50 units; 100 units) and myxoploids (50 units; 100 units) has been investigated. Homozygous lines were created by stabilizing the genome ploidy of haploid and myxoploid micro sprouts during III–IV passages without the use of colchicine. Technologies of rooting in the open ground for use in the breeding process of sugar beets have been improved.

Pollen fertility differences in the progenies obtained from a cross between eggplant (Solanum melongena L.) as a seed parent and eggplant cytoplasmic substitution lines as pollen parents

To the best of our knowledge, there is no report about pollen fertility of the progenies developed using eggplant (Solanum melongena L.) as a seed parent and eggplant cytoplasmic substitution lines as pollen parents. Pollen fertility of these progenies is very important to use as restorer line in the eggplant’s hybrid breeding program. In this study, pollen fertility was investigated for the progenies which were produced using S. melongena ‘Uttara’ as a seed parent and the eggplant cytoplasmic substitution lines as pollen parents. To assess pollen fertility, pollen stainability and in vitro germination ability were investigated. Although the nuclear and the cytoplasmic genome of the progenies were almost identical to eggplant ‘Uttara’, a clear difference was observed in the pollen fertility due to the difference in the pollen parents having different wild Solanum cytoplasms. The progenies produced using the functional cytoplasmic male sterile (CMS) lines as a pollen parent, whose cytoplasm donor were S. kurzii, S. violaceum and S. virginianum, showed pollen release type and high pollen fertility almost equal to eggplant ‘Uttara’. It is considered that the characteristics of these progenies were almost the same as eggplant. On the other hand, the progenies that produced using the fertility restored lines of the pollen non-formation type CMS lines as a pollen parent, whose cytoplasm donors were S. aethiopicum, S. anguivi and S. grandifolium, showed pollen release type and low pollen fertility, i.e., pollen staining ability was about 54% and pollen germination ability were about 35%. It is considered that the cause of this low pollen fertility was the incompatibility between the eggplant cytoplasm and the eggplant nuclear genome, which seems to be modified in the process of continuous backcrossing under the wild Solanum cytoplasms. It is suggested that complete nuclear substitution is difficult by continuous backcrossing with eggplant in the alloplasmic lines with S. aethiopicum, S. anguivi and S. grandifolium cytoplasm donors. Incompatibility between the normal eggplant cytoplasm and the modified eggplant nuclear genomes of the alloplasmic lines with S. aethiopicum, S. anguivi and S. grandifolium cytoplasms might be a cause for the low pollen fertility of the investigated progenies

Improvement of the technology of obtaining stable (di)haploid regenerants from embryonic culture of apomictic sugar beet (Beta vulgaris) breeding material without the use of colchicine

Aim. To evaluate the effi ciency of inducing generative, reduced parthenogenesis and to better use the differentiating potential of the embryo culture under apomictic seed production in selection materials of sugar beet with cytoplasmic male sterility (CMS), and B) to isolate homozygous lines (dihaploids) without the use of polyploidizing substances. Methods. Apomictic (agamosper- mous) seed production in apocarpous pollen sterile lines from B. vulgaris subsp. vulgaris var. altissima (sugar beet) using classi- cal so-called Owen sterile cytoplasm and sterile cytoplasm from Beta maritimа and Beta patula as sources, was conducted under pollen free conditions and spatial isolation in the greenhouse breeding complex of the Yaltushkivska experimental breeding station (Yaltushki, Ukraine). The specifi cities of embryonic development of apomictic embryos were studied with the purpose of effi cient regulation of the induction of explants in vitro as donors of the culture of immature embryos. Fluorescent fl ow cytophotometry in combination with the computer program of the Partec Ploidy Analyser PA-2 (Partec GmbH, Germany, now Sysmex), were used to determine the degree of ploidy, enabling the selection of haploid and dihaploid lines in vitro. A genetic method was developed using the expression of morphological marker indices of nuclear genes of anthocyanin coloring (R+ r–) of regenerant plants in vitro and ploidy determination for differentiation by generative (reduced) parthenogenesis. The sampling technique that took into account the hormonal composition of cultural media and the level of genome ploidy, sample frequency and statistical analysis of the results was determined using the appropriate statistics; the percentage of regenerants, induced by different types of morpho- genesis and ploidy in vitro, was determined along with the measurement error to control the accuracy of the selected sampling (number of seed embryos). Results. The selected cultural medium No. 3, based on the basal medium according to Gamberg et al., 1968 (21), contained 6 BAP – 2 mg/l, 2.4 D – 0.5 mg/l, gibberellic acid – 0.1 mg/l, which ensured a success rate of 4.4 to 23.3 % of direct regeneration of shoots from the embryo culture, depending on the genotype of donors, and 4–10 % for induction and proliferation of callus. In ten experimental numbers of alloplasmic lines of sugar beet, the incidence of haploids and mixoploids among the regenerants from the embryo culture fl uctuated within the wide range of 14.8 – 62.2 % and exceeded the indices, ob- tained by other known methods of haploid parthenogamy, which had the values of 3.79 – 6.25 %. Conclusions. The homozygous lines and dihaploids were determined and set apart/stabilized in the process of micropropagation, where the differentiation of clones was made on the basis of total DNA content in interphase nuclei, using information of histograms generated in fl uorescent fl ow cytometry with the Partec Ploidy Analyser PA-II instrumentation. The medium, based on macro- and microsalts according to Gamberg et al., 1968 (21) was found to be the most effi cient; it ensured at least partially successful direct regeneration in the culture of embryos within the range of 4.40 ± 1.29 to 23.3 ± 3.45 %. The success of direct regeneration of apomictic material depended on the composition of the cultural medium used fi rst and foremost, and to a lesser extent on the stages of embryogenesis from day 12 till day 32, differentiated by the fi xation period for seed embryos starting from the beginning of fl owering. Homozygous lines were created without polyploid-inducing substances due to spontaneous transfer of some cells of haploid regenerant plants to a higher level of ploidy, that can be used in the breeding of sugar beet. Genetic determination of apomictic seed reproduction in alloplasmic lines and pollen free lines of sugar beet and the technologies of inducing dihaploids allow reducing the period of inzucht-crossing considerably to obtain homozygous lines, creating unique material for chromosome engineering and marker-oriented selection with target combinations of genes in homozygous state.

Alloplasmic recombinant lines (H. vulgare)-T. aestivum with 1RS.1BL translocation: initial genotypes for production of common wheat varieties

Alloplasmic lines are formed when the cytoplasm of one species is replaced by the cytoplasm of another as a result of repeated recurrent crosses of wide hybrids with the paternal genotype. Since the cytoplasm replacement results in new intergenomic interactions between a nucleus and cytoplasm leading to variability of plant characteristics, alloplasmic lines with restored fertility can be an additional source of biodiversity of cultivated plants. Earlier, recombinant alloplasmic lines (H. vulgare)-T. aestivumdesignated as L-17(1)–L-17(37) were formed from a plant with partially restored fertility of the BC3 generation of barley-wheat hybridH. vulgare(cv. Nepolegayushchii) ×T. aestivum(cv. Saratovskaya 29). This male-sterile hybrid was consistently backcrossed with wheat varieties Mironovskaya 808 (twice) and Saratovskaya 29, and Mironovskaya 808 had a positive impact on the restoration of fertility. This paper presents the results of investigation into a group of recombinant alloplasmic lines (L-17F4), as well as into doubled haploids (DH) lines – alloplasmic DH-17-lines obtained from anther culture of alloplasmic lines (L-17F2). The most productive of these lines were used as initial breeding genotypes. Hybrid form Lutescens 311/00-22 developed from the crossing of the alloplasmic DH(1)-17 line (as maternal genotype) with euplasmic line Com37 (CIMMYT), the source of the 1RS.1BL wheat-rye translocation, proved to be successful for breeding. The presence of the 1RS.1BL translocation in the genome of the Lutescens 311/00-22 form and the L-311(1)–L-311(6) alloplasmic lines isolated from it did not lead to a decrease of fertility or sterility in the plants. This indicates that the chromosome of the 1BS wheat does not carry the gene(s) that determine the restoration of fertility in the studied (H. vulgare)-T. aestivumalloplasmic lines. Alloplasmic lines L-311(1)–L-311(6) showed their advantage in comparison with the standard varieties for resistance to leaf and stem rust, yield, and grain quality. The breeding tests performed at Omsk Agricultural Scientific Center, Agrocomplex “Kurgansemena”, Federal State Unitary Enterprise “Ishimskoe” (Tyumen Region), using alloplasmic lines L-311(5), L-311(4) and L-311(6) resulted in varieties of spring common wheat Sigma, Uralosibirskaya 2 and Ishimskaya 11, respectively.